Use of pasteurized akkermansia for treating metabolic disorders

ABSTRACT

The present invention relates to pasteurized  Akkermansia muciniphila  or fragments thereof for treating a metabolic disorder in a subject in need thereof. The present invention also relates to a composition, a pharmaceutical composition and a medicament comprising pasteurized  Akkermansia muciniphila  or fragments thereof for treating a metabolic disorder. The present invention also relates to the use of pasteurized  Akkermansia muciniphila  or fragments thereof for promoting weight loss in a subject in need thereof.

FIELD OF INVENTION

The present invention relates to the treatment of metabolic disorders,such as, for example, metabolic disorders related to overweight andobesity, such as, for example, Diabetes Mellitus or high cholesterol.The present invention more specifically relates to a compositioncomprising pasteurized Akkermansia spp. or fragments thereof fortreating a metabolic disorder.

BACKGROUND OF INVENTION

Obesity is a worldwide problem, with an estimated number of obese adultsof about 600 million. This epidemic of obesity is correlated with agreat increase in the prevalence of obesity-related disorders, such as,for example, diabetes, hypertension, cardiac pathologies and liverdiseases. Due to these highly disabling pathologies, obesity iscurrently considered in western countries as one of the most importantpublic health problems. There is thus a real need of compositions andmethods for treating or preventing obesity and/or obesity-relateddisorders.

Obesity and obesity-related diseases are associated with (i) metabolicdysfunctions (with an impact on glucose homeostasis and lipid metabolismfor example); (ii) low grade inflammatory state associated to higherblood lipopolysaccharides (LPS) levels (also referred as metabolicendotoxemia); and (iii) impaired gut barrier function (i.e. increasedgut permeability) leading to translocation of bacteria and/ormicroorganisms components into organs such as the liver or the adiposetissue. In order to treat obesity, impact on at least one, preferably 2and more preferably 3 of these 3 factors is thus needed. These phenomena(i.e., intestinal inflammation, LPS and bacterial translocation) arealso observed during inflammatory bowel diseases, such as for instanceCrohn's diseases, colitis, ulcerative colitis, intestinal pain (e.g.,colic) and other intestinal inflammatory diseases. Interestingly, bothinflammatory bowel diseases and obesity-related diseases are associatedwith changes in the gut microbiota composition. Thus, reinforcing thegut barrier function is one of the major issues.

The human gut is colonized by a diverse, complex and dynamic communityof microbes representing over 1000 different species, which continuouslyinteract with the host (Zoetendal et al., 2008. Gut. 57(11):1605-1615;Rajilic-Stojanavic and de Vos, 2014. FEMS Microbiol. Rev. 38:996-1047).The homeostasis of the gut microbiota is dependent on hostcharacteristics (age, gender, genetic background . . . ) andenvironmental conditions (stress, drugs, gastrointestinal surgery,infectious and toxic agents . . . ), but also on the day-to-day dietarychanges.

It has been recently acknowledged that the intestinal microbiota isinvolved in a number of brain disorders, such as anxiety, autism (Hsiaoet al., 2013. Cell. 155(7):1451-1463), Parkinson's disease (Scheperjanset al., 2015. Mov. Disord. 30(3):350-8), Alzheimer's disease (Harach etal., 2015. arXiv:1509.02273), and in multiple sclerosis (Berer et al.,2011. Nature. 479(7374):538-41).

Gut microbiota imbalance was also shown to be a risk factor for thedevelopment of cancers such as colorectal cancer (Zitvogel et al., 2015.Sci. Transl. Med. 7(271):271ps1; Louis et al., 2014. Nat. Rev.Microbiol. 12(10):661-72).

Growing evidences also support the role of gut microbiota in thedevelopment of obesity and related disorders (Delzenne & Cani, 2011.Annu. Rev. Nutr. 31:15-31) and intestinal inflammation (Wlodarska etal., 2015. Cell Host Microbe. 17(5):577-91), or intestinal pain (forexample, babies' colic) (de Weerth et al., 2013. Pediatrics. 131:e550).In all these cases (obesity, intestinal inflammation, colic), dysbiosisof the microbiota can further disrupt the crosstalk between organs andthe integrity of the intestinal barrier leading to symptoms.

Therefore, treatment with products that target the gut microbiotaappeared as promising therapeutic tools for treating obesity and relateddisorders. These products may consist of living microbes, such as in thecase of most probiotics, or contain dead microbes or fragments thereof.In addition, these products may comprise substrates that are used by thegut microbiota, such as in the case of prebiotics, or contain compoundsthat change the balance of the intestinal microbiota, such as specificantimicrobial compounds.

For example, WO 2008/076696 describes the gut microbiota as atherapeutic target for treating obesity and related disorders. WO2008/076696 specifically describes methods for altering the abundance ofBacteroidetes and/or Firmicutes in the gut of a subject, byadministering antibiotics and/or probiotics to the subject.

Moreover, EP 2 030 623 relates to the prevention and/or treatment ofmetabolic disorders, such as, for example, obesity related disorders, byregulating the amount of Enterobacteria in the gut. EP 2 030 623discloses reducing the amount of Enterobacteria in the gut byadministering probiotic bacteria, such as, for example, Bifidobacterium,Lactococcus, Streptococcus, Enterococcus or Lactobacillus.

The patent application US 2012/083514 relates to infant cerealscomprising non-replicating probiotic micro-organisms. US 2012/083514describes three types of heat treatment: 140° C. for 15 seconds (ultrahigh temperature); 74° C., 90° C. and 120° C. for 15 seconds (hightemperature short time); and 85° C. for 20 minutes (long time lowtemperature). However, it is shown in this patent application US2012/083514 that the ratio IL12/IL10 strongly increases in bacteriasubmitted to heat treatment at 85° C. for 20 minutes. IL12 is aproinflammatory cytokine, while IL10 is an anti-inflammatory cytokine.US 2012/083514 thus demonstrates that a heat treatment at 85° C. for 20minutes increases the inflammatory state of the subject and is thereforenot recommended for treating inflammatory disorders. Meanwhile, US2012/083514 demonstrates that bacteria have to be heated for a veryshort time (15 seconds) to present an anti-inflammatory profile.

Furthermore, the Applicant described that the gut microbiota is modifiedin prebiotic-treated obese mice (Everard et al., 2011 November Diabetes.60(11):2775-86). Moreover, prebiotics (1) improve glucose and lipidmetabolisms in obese and diabetic mice, (2) reduce plasma LPS andimprove gut barrier function (e.g. reduction of inflammation) in obesemice, (3) induce an increased enteroendocrine L-cell number in obese anddiabetic mice, and (4) improve leptin sensitivity and glucosehomeostasis in diet-induced obese and diabetic mice.

The Applicant also described the use of Akkermansia muciniphila orfragments thereof for treating obesity and related disorders (WO2014/076246). Moreover, the Applicant also disclosed a reduced abundanceof Akkermansia muciniphila in the gut of patients suffering fromulcerative colitis (Rajilić-Stojanović M et al., 2013 March Inflamm.Bowel Dis. 19(3):481-8). In Crohn's disease mainly butyrate-producingbacteria were found to be depleted (Wlodarska et al., 2015. Cell HostMicrobe. 17(5):577-91). However, it was shown that Akkermansiamuciniphila, which produces the short chain fatty acids propionate andacetate, can also give rise to trophic chains that produce butyrate asend product from mucus. Butyrate is known to reduce pain sensation inthe gut and, like acetate and propionate, is known to show immunesignaling. Finally, it has been shown that addition of Akkermansiamuciniphila increases the barrier function in a human cell line(Reunanen et al., 2015. Appl. Environ. Microbiol. 81(11):3655-62).Hence, it is very likely that Akkermansia muciniphila and its productsmay reduce intestinal pain and inflammation as well as reinforce the gutbarrier in healthy human as well as in patients suffering fromintestinal inflammatory diseases. This may not only apply to adults butalso to infants, as reduced butyrate producers were associated withexcessive crying in baby colic and atopic diseases in young infants (deWeerth et al., 2013. Gut Microbes. 4(5):416-21; Nylund et al., 2015.Allergy. 70(2):241-4).

However, here, the Applicant surprisingly showed that administration ofpasteurized Akkermansia muciniphila is more efficient thannon-pasteurized Akkermansia muciniphila to increase barrier function andtreat metabolic dysfunctions associated with obesity and relateddisorders. The present invention thus relates to the use of pasteurizedAkkermansia muciniphila or fragments thereof to increase barrierfunction and treating obesity and related disorders.

SUMMARY

The present invention relates to Akkermansia muciniphila or fragmentsthereof for use in treating a metabolic disorder in a subject in needthereof, wherein Akkermansia muciniphila is pasteurized. In oneparticular embodiment, Akkermansia muciniphila or fragments thereof isfor use in treating obesity.

In one embodiment, Akkermansia muciniphila or fragments thereof is foruse in treating a metabolic disorder, wherein said metabolic disorder isselected from the group comprising metabolic syndrome;insulin-deficiency or insulin-resistance related disorders; DiabetesMellitus including Type 2 Diabetes; glucose intolerance; abnormal lipidmetabolism; atherosclerosis; hypertension; pre-eclampsia; cardiacpathology; stroke; non-alcoholic fatty liver disease; hyperglycemia;hepatic steatosis; liver diseases including fibrosis associated withobesity and abnormal liver functions, more particularly changes in bileproduction and immunity; dyslipidemia; dysfunction of the immune systemassociated with overweight and obesity; inflammatory, immune and barrierfunction diseases, including inflammatory bowel disease, moreparticularly Crohn's disease and ulcerative colitis, and irritable bowelsyndrome; cardiovascular diseases; high cholesterol; elevatedtriglycerides; asthma; sleep apnea; osteoarthritis; neuro-degeneration;gallbladder disease; syndrome X; atherogenic dyslipidemia and cancer.

The present invention also relates to Akkermansia muciniphila orfragments thereof for increasing energy expenditure of a subject,preferably without impacting the food intake of said subject, whereinAkkermansia muciniphila is pasteurized.

Another object of the invention is Akkermansia muciniphila or fragmentsthereof for increasing satiety in a subject, wherein Akkermansiamuciniphila is pasteurized.

In one embodiment, Akkermansia muciniphila or fragments thereof for useas described hereinabove is orally administered.

In one embodiment, Akkermansia muciniphila or fragments thereof for useas described hereinabove is administered to the subject in an amountranging from about 1.10⁴ to about 1.10¹² cells, more preferably fromabout 1.10⁵ to about 1.10¹¹ cells, and even more preferably from about1.10⁶ to about 1.10¹⁰ cells.

In one embodiment of the invention, Akkermansia muciniphila or fragmentsthereof for use as described hereinabove is administered at least threetimes a week.

In one embodiment, Akkermansia muciniphila or fragments thereof for useas described hereinabove is co-administered with another probioticstrain and/or another bacteria and/or microorganisms with beneficialeffects and/or with one or more prebiotics.

The present invention also relates to a composition for use for treatinga metabolic disorder or for increasing energy expenditure of a subjector for increasing satiety in a subject comprising Akkermansiamuciniphila or fragments thereof as described hereinabove in associationwith an excipient.

In one embodiment, said composition for use is a nutritionalcomposition. In one embodiment, said composition for use is orallyadministered.

Another object of the invention is a pharmaceutical composition fortreating a metabolic disorder or for increasing energy expenditure of asubject or for increasing satiety in a subject comprising Akkermansiamuciniphila or fragments thereof as described hereinabove in associationwith a pharmaceutically acceptable vehicle.

The present invention further relates to a medicament for treating ametabolic disorder or for increasing energy expenditure of a subject orfor increasing satiety in a subject comprising Akkermansia muciniphilaor fragments thereof as described hereinabove.

The present invention also relates to the use of Akkermansia muciniphilaor fragments thereof for promoting weight loss in a subject in needthereof, wherein Akkermansia muciniphila is pasteurized.

Another object of the invention is a cosmetic composition comprisingAkkermansia muciniphila or fragments thereof for promoting weight lossin a subject in need thereof, wherein Akkermansia muciniphila ispasteurized.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “Treatment” means preventing (i.e. keeping from happening),        reducing or alleviating at least one adverse effect or symptom        of a disease, disorder or condition. This term thus refers to        both therapeutic treatment and prophylactic or preventative        measures; wherein the object is to prevent or slow down (lessen)        the targeted pathologic condition or disorder. In one embodiment        of the invention, those in need of treatment include those        already with the disorder as well as those prone to have the        disorder or those in whom the disorder is to be prevented.    -   “Effective amount” refers to level or amount of agent that is        aimed at, without causing significant negative or adverse side        effects to the target, (1) delaying or preventing the onset of a        metabolic disorder; (2) slowing down or stopping the        progression, aggravation, or deterioration of one or more        symptoms of the metabolic disorder; (3) bringing about        ameliorations of the symptoms of the metabolic disorder; (4)        reducing the severity or incidence of the metabolic        disorder; (5) curing the metabolic disorder; or (6) restoring        the normal amount and/or proportion of Akkermansia muciniphila        in the gut of the subject to be treated. An effective amount may        be administered prior to the onset of a metabolic disorder, for        a prophylactic or preventive action. Alternatively or        additionally, the effective amount may be administered after        initiation of the metabolic disorder, for a therapeutic action.    -   “Akkermansia muciniphila” refers to the mucin-degrading bacteria        identified by Derrien (Derrien et al., 2004. Int. J. Syst. Evol.        Microbiol. 54:1469-1476). Cells arc oval-shaped, non-motile and        stain Gram-negative. Akkermansia muciniphila may also be        referred as Akkermansia spp. or Akkermansia-like bacteria. It        belongs to the Chlamydiae/Verrucomicrobia group; Verrucomicrobia        phylum. If the taxonomy should change, the skilled artisan would        know how to adapt the changes in the taxonomy to deduce the        strains that could be used in the present invention. Moreover,        the complete genome of Akkermansia muciniphila has been        determined by the Applicant (van Passel et al., 2011. PLoS One.        6(3):e16876). It is generally accepted that strains with a        nucleotide similarity as experimentally determined by DNA-DNA        hybridization of about 70% can be considered as the same        species—this corresponds to an average nucleotide identity (ANI)        of approximately 95% (Goris et al., 2007. Int. J. Syst. Evol.        Microbiol. 57:81-91).    -   “Pasteurized Akkermansia muciniphila” refers to Akkermansia        muciniphila submitted to a heating treatment. In one embodiment,        pasteurized Akkermansia muciniphila refers to Akkermansia        muciniphila which was heated at a temperature from 50° C. to        100° C. for at least 10 minutes.    -   “Probiotics” refers to microbial cell preparations (such as, for        example, living microbial cells) which, when administered in an        effective amount, provide a beneficial effect on the health or        well-being of a subject. By definition, all probiotics have a        proven non-pathogenic character. In one embodiment, these health        benefits are associated with improving the balance of human or        animal microbiota in the gastrointestinal tract, and/or        restoring normal microbiota.    -   “Prebiotic” refers to a substance, such as, for example, a        substance which may not be digested by humans, but which        modulates composition and/or activity of the gut microbiota        through its metabolization by microorganisms in the gut, thus        conferring a beneficial physiological effect on the host.    -   “Subject” refers to an animal, preferably a mammal, more        preferably a human. In one embodiment, the subject is a male. In        another embodiment, the subject is a female. In one embodiment        of the invention, a subject may also refer to a pet, such as,        for example, a dog, a cat, a guinea pig, a hamster, a rat, a        mouse, a ferret, a rabbit and the like.    -   “Overweight” refers to a subject situation wherein said subject        has a Body Mass Index (BMI) ranging from 25 to 30. As used        herein, BMI is defined as the individual's body mass (in kg)        divided by the square of his/her height (in meter).    -   “Obesity” refers to a subject situation wherein said subject has        a BMI superior or equal to 30.    -   “About” preceding a figure means plus or less 20%, preferably        10% of the value of said figure.    -   “Fragment” may refer to cellular components, metabolites,        secreted molecules and compounds resulting from the metabolism        of pasteurized Akkermansia muciniphila and the like. Fragments        may be obtained, for example, by recovering the supernatant of a        culture of Akkermansia muciniphila after pasteurization or by        extracting cell components or cell fractions, metabolites or        secreted compounds from a culture of Akkermansia muciniphila        after pasteurization. The term fragment may also refer to a        degradation product. A fragment may correspond to a component in        the isolated form or to any mixture of one or more components        derived from pasteurized Akkermansia muciniphila. In one        embodiment, a fragment may correspond to one or more of such a        components present in pasteurized Akkermansia muciniphila that        is produced in another way, such as using recombinant DNA        technology, in a microbial host or in any other (bio)synthetic        process.    -   “Metabolic disorder” refers to disorders, diseases and        conditions caused or characterized by abnormal weight gain,        energy use or consumption, altered responses to ingested or        endogenous nutrients, energy sources, hormones or other        signaling molecules within the body or altered metabolism of        carbohydrates, lipids, proteins, nucleic acids or a combination        thereof. A metabolic disorder may be associated with either a        deficiency or an excess in a metabolic pathway resulting in an        imbalance in metabolism of carbohydrates, lipids, proteins        and/or nucleic acids. Examples of metabolic disorders include,        but are not limited to, metabolic syndrome, insulin-deficiency        or insulin-resistance related disorders, Diabetes Mellitus (such        as, for example, Type 2 Diabetes), glucose intolerance, abnormal        lipid metabolism, atherosclerosis, hypertension, pre-eclampsia,        cardiac pathology, stroke, non-alcoholic fatty liver disease,        hyperglycemia, hepatic steatosis from different etiology,        dyslipidemia, dysfunction of the immune system associated with        overweight and obesity, cardiovascular diseases, high        cholesterol, elevated triglycerides, asthma, sleep apnea,        osteoarthritis, neuro-degeneration, gallbladder disease,        syndrome X, inflammatory and immune disorders, atherogenic        dyslipidemia and cancer.

DETAILED DESCRIPTION

The Applicant herein shows that the beneficial effects on metabolismobserved after pasteurized Akkermansia muciniphila administration aremore important than after non-pasteurized Akkermansia muciniphilaadministration (see Examples).

Therefore, this invention relates to pasteurized Akkermansia muciniphilaor a fragment thereof for treating, or for use in treating, metabolicdisorders in a subject in need thereof.

As used herein, a metabolic disorder is a disorder related to an alteredmetabolic homeostasis, such as, for example, an altered glucose or lipidhomeostasis.

In one embodiment of the invention, said metabolic disorder is obesity.

Examples of other metabolic disorders include, but are not limited to,metabolic syndrome, insulin-deficiency or insulin-resistance relateddisorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes),glucose intolerance, abnormal lipid metabolism, atherosclerosis,hypertension, pre-eclampsia, cardiac pathology, stroke, non-alcoholicfatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia,dysfunction of the immune system associated with overweight and obesity,liver diseases (such as, for example, fibrosis associated with obesity,or abnormal liver functions, including changes in bile production,immunity, and the like), inflammatory, immune and barrier functiondiseases (such as, for example, inflammatory bowel disease, includingCrohn's disease and ulcerative colitis, and irritable bowel syndrome),cardiovascular diseases, high cholesterol, elevated triglycerides,asthma, sleep apnea, osteoarthritis, neuro-degeneration, gallbladderdisease, syndrome X, inflammatory and immune disorders, atherogenicdyslipidemia and cancer.

In another embodiment, said metabolic disorder is an overweight and/orobesity related metabolic disorder, i.e. a metabolic disorder that maybe associated to or caused by overweight and/or obesity. Examples ofoverweight and/or obesity related metabolic disorders include, but arenot limited to metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders, Diabetes Mellitus (such as, forexample, Type 2 Diabetes), glucose intolerance, abnormal lipidmetabolism, atherosclerosis, hypertension, cardiac pathology, stroke,non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis,dyslipidemia, dysfunction of the immune system associated withoverweight and obesity, cardiovascular diseases, high cholesterol,elevated triglycerides, asthma, sleep apnea, osteoarthritis,neuro-degeneration, gallbladder disease, syndrome X, inflammatory andimmune disorders, atherogenic dyslipidemia and cancer.

In one embodiment, said metabolic disorder is Diabetes Mellitus,preferably Type 2 Diabetes. In another embodiment, said metabolicdisorder is hypercholesterolemia (also known as high cholesterol). Inone embodiment, hypercholesterolemia corresponds to a plasma cholesterolconcentration superior or equal to 2 g/L or 5 mmol/L. In anotherembodiment, hypercholesterolemia corresponds to a ratio plasmaconcentration of total cholesterol:plasma concentration of HDL (highdensity lipoprotein cholesterol) superior or equal to 4.5:1, preferably5:1.

As used herein, the term “pasteurized Akkermansia muciniphila” meansAkkermansia muciniphila submitted to heating. In one embodiment, thepasteurized Akkermansia muciniphila of the invention was heated at atemperature from 50° C. to 100° C., preferably from 60° C. to 95° C.,more preferably from 70° C. to 90° C. In one embodiment, the pasteurizedAkkermansia muciniphila of the invention was heated at a temperature of50, 51, 52, 53, 54, 55, 56, 57, 58 or 59° C. In another embodiment, thepasteurized Akkermansia muciniphila of the invention was heated at atemperature of 60, 61, 62, 63, 64, 65, 66, 67, 68 or 69° C. In yetanother embodiment, the pasteurized Akkermansia muciniphila of theinvention was heated at a temperature of 70, 71, 72, 73, 74, 75, 76, 77,78 or 79° C. In yet another embodiment, the pasteurized Akkermansiamuciniphila of the invention was heated at a temperature of 80, 81, 82,83, 84, 85, 86, 87, 88 or 89° C. In yet another embodiment, thepasteurized Akkermansia muciniphila of the invention was heated at atemperature of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99° C. or 100° C.

In one embodiment, the pasteurized Akkermansia muciniphila of theinvention was not heated at a temperature superior to 100° C. In aparticular embodiment, the pasteurized Akkermansia muciniphila of theinvention was not heated at an ultra-high temperature, such as forexample at a temperature of 110° C. to 140° C. In one embodiment, thepasteurized Akkermansia muciniphila of the invention was not heated at atemperature superior to 90° C. Accordingly, in one embodiment of theinvention, Akkermansia muciniphila was not sterilized. Sterilization isa treatment intended to destroy, kill or inactivate all life forms andother biological agents. This includes microorganisms and their sporesas well as viruses and prions. Unlike sterilization, pasteurization isnot intended to kill all microorganisms but is usually applied to foodwith the aim to reduce the number of viable pathogens.

In one embodiment of the invention, the pasteurized Akkermansiamuciniphila was heated for at least 10 minutes. In another embodiment ofthe invention, the pasteurized Akkermansia muciniphila was heated for atleast 15, 20, 25, 30, 35 or 45 minutes. In one embodiment, thepasteurized Akkermansia muciniphila of the invention was heated for aperiod from 10 to 45 minutes.

In one embodiment, the pasteurized Akkermansia muciniphila was notheated for a short time. In a particular embodiment, the pasteurizedAkkermansia muciniphila was not heated for a time of 1 to 30 seconds, of1 to 60 seconds, of 1 to 90 seconds or of 1 to 120 seconds. In apreferred embodiment, the pasteurized Akkermansia muciniphila was notheated for a time of less than 1 minute, preferably for a time of lessthan 5, 6, 7, 8, or 9 minutes.

In one embodiment, the pasteurized Akkermansia muciniphila was heated ata temperature from 50° C. to 100° C. for at least 10 minutes. In aparticular embodiment, the pasteurized Akkermansia muciniphila of theinvention was heated to 60° C. for 20 or 30 minutes. In anotherparticular embodiment, the pasteurized Akkermansia muciniphila of theinvention was heated to 70° C. for 20 or 30 minutes. In anotherparticular embodiment, the pasteurized Akkermansia muciniphila of theinvention was heated to 80° C. for 20 or 30 minutes. In anotherparticular embodiment, the pasteurized Akkermansia muciniphila of theinvention was heated to 90° C. for 20 or 30 minutes.

In a particular embodiment, the pasteurized Akkermansia muciniphila wasnot heated at a temperature superior to 110° C. for about 1 to 120seconds. In another particular embodiment, the pasteurized Akkermansiamuciniphila was not heated at a temperature superior to 100° C. forabout 1 to 120 seconds. In another particular embodiment, thepasteurized Akkermansia muciniphila was not heated at a temperaturesuperior to 90° C. for about 1 to 120 seconds.

According to one embodiment, pasteurized Akkermansia muciniphila of theinvention are non-viable cells. As used herein, “non-viable cells” meanscells that are not able to proliferate. Measurement of cell viabilityand proliferation may be any method known to one skilled in the art. Forexample, cell viability and proliferation may be assessed by spreading asolution containing pasteurized Akkermansia muciniphila across a petridish or using any other culture methods and counting the number ofclones or optical density after a determined time of incubation inoptimal growth conditions. Moreover, it is also possible to determinethe number of cells, including viable as well as non-viable cells atleast as the integrity of the cells is not compromised, by microscopicobservation. While phase-contrast microscopy is a well-known method todo so, the microbial cells can be further visualized by specificstaining with dyes, fluorescent probes or antibodies. This allowsfacilitation of microscopic observations while the number of stainedcells can be also be counted by flow cytometry. Examples to visualize orcounts cells of Akkermansia muciniphila have been provided by Derrien etal. (2008. Appl. Environ. Microbiol. 74:1646-8), Derrien et al. (2011.Frontiers Microbiol. 2:166-175) or Reunanen et al. (2015. Appl. Environ.Microbiol. 81(11):3655-62).

In one embodiment, pasteurized Akkermansia muciniphila or a fragmentthereof is substantially purified. As used herein, the term“substantially purified” means that pasteurized Akkermansia muciniphilaor fragment thereof is comprised in a sample wherein it represents atleast about 50%, preferably at least about 60, 70, 80, 85, 90, 95, 99%or more of the bacterial strains or fragment thereof of said sample.

The present invention also relates to a composition comprising aneffective amount of pasteurized Akkermansia muciniphila or a fragmentthereof for treating, or for use in treating, a metabolic disorder.

In one embodiment of the invention, the effective amount of pasteurizedAkkermansia muciniphila corresponds to the amount of the bacteriasufficient for restoring a normal amount and/or proportion ofAkkermansia muciniphila within the gut of the subject. In one embodimentof the invention, the normal amount and/or proportion of Akkermansiamuciniphila corresponds to the amount, and/or to the proportion ofAkkermansia muciniphila present in the gut of a healthy subject.

As used herein, the term “healthy subject” is used to define a subjectwhich is not affected by the disease to be treated. For example, ifpasteurized Akkermansia muciniphila or a fragment thereof is used fortreating obesity, the healthy subject is not affected by obesity.Preferably, the healthy subject shares common characteristics with thesubject to be treated, such as, for example, same gender, age, sex,diet, drugs intake or geolocation.

In one embodiment of the invention, the normal proportion of Akkermansiamuciniphila in the gut ranges from about 0.1% to about 10% (in number ofAkkermansia muciniphila cells to the total number of bacteria cells ofthe gut), preferably from about 0.3% to about 5%, more preferably fromabout 1% to about 3%.

In one embodiment, the effective amount of pasteurized Akkermansiamuciniphila of the invention corresponds to an amount of Akkermansiamuciniphila before the step of pasteurization ranging from about 1.10²to about 1.10¹⁵ cfu, preferably from about 1.10⁴ to about 1.10¹² cfu,more preferably from about 1.10⁵ to about 1.10¹⁰ cfu, and even morepreferably from about 1.10⁶ to about 1.10⁹ cfu, wherein cfu stands for“colony forming unit”.

In another embodiment of the invention, the effective amount ofpasteurized Akkermansia muciniphila corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging fromabout 1.10⁶ to about 1.10¹⁰ cfu, preferably from about 1.10⁸ to about1.10¹⁰ cfu, more preferably from about 1.10⁹ to about 1.10¹⁰ cfu.

In another embodiment of the invention, the effective amount ofpasteurized Akkermansia muciniphila corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging fromabout 1.10⁶ to about 1.10¹¹ cfu, preferably from about 1.10⁸ to about1.10¹¹ cfu, more preferably from about 1.10¹⁰ to about 1.10¹¹ cfu.

In one embodiment, the effective amount of pasteurized Akkermansiamuciniphila of the invention ranges from about 1.10² to about 1.10¹⁵cells, preferably from about 1.10⁴ to about 1.10¹² cells, morepreferably from about 1.10⁵ to about 1.10¹⁰ cells, and even morepreferably from about 1.10⁶ to about 1.10⁹ cells.

In another embodiment of the invention, the effective amount ofpasteurized Akkermansia muciniphila ranges from about 1.10⁶ to about1.10¹⁰ cells, preferably from about 1.10⁸ to about 1.10¹⁰ cells, morepreferably from about 1.10⁹ to about 1.10¹⁰ cells.

In another embodiment of the invention, the effective amount ofpasteurized Akkermansia muciniphila ranges from about 1.10⁶ to about1.10¹¹ cells, preferably from about 1.10⁸ to about 1.10¹¹ cells, morepreferably from about 1.10¹⁰ to about 1.10¹¹ cells.

In one embodiment of the invention, the effective amount of a fragmentof pasteurized Akkermansia muciniphila corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging fromfragments derived from about 1.10² to about 1.10¹⁵ cfu, preferably fromabout 1.10⁴ to about 1.10¹² cfu, more preferably from about 1.10⁵ toabout 1.10¹⁰ cfu, and even more preferably from about 1.10⁶ to about1.10⁹ cfu, wherein cfu stands for “colony forming unit”.

In another embodiment of the invention, the effective amount of afragment of pasteurized Akkermansia muciniphila corresponds to an amountof Akkermansia muciniphila before the step of pasteurization rangingfrom fragments derived from about 1.10⁶ to about 1.10¹⁰ cfu, preferablyfrom about 1.10⁸ to about 1.10¹⁰ cfu, more preferably from about 1.10⁹to about 1.10¹⁰ cfu.

In another embodiment of the invention, the effective amount of afragment of pasteurized Akkermansia muciniphila corresponds to an amountof Akkermansia muciniphila before the step of pasteurization rangingfrom fragments derived from about 1.10⁶ to about 1.10¹¹ cfu, preferablyfrom about 1.10⁸ to about 1.10¹⁰ cfu, more preferably from about 1.10¹⁰to about 1.10¹¹ cfu.

In one embodiment of the invention, the effective amount of a fragmentof pasteurized Akkermansia muciniphila ranges from fragments derivedfrom about 1.10² to about 1.10¹⁵ cells, preferably from about 1.10⁴ toabout 1.10¹² cells, more preferably from about 1.10⁵ to about 1.10¹⁰cells, and even more preferably from about 1.10⁶ to about 1.10⁹ cells.In another embodiment of the invention, the effective amount of afragment of pasteurized Akkermansia muciniphila ranges from fragmentsderived from about 1.10⁶ to about 1.10¹⁰ cells, preferably from about1.10⁸ to about 1.10¹⁰ cells, more preferably from about 1.10⁹ to about1.10¹⁰ cells.

In another embodiment of the invention, the effective amount of afragment of pasteurized Akkermansia muciniphila ranges from fragmentsderived from about 1.10⁶ to about 1.10¹¹ cells, preferably from about1.10⁸ to about 1.10¹¹ cells, more preferably from about 1.10¹⁰ to about1.10¹¹ cells.

In one embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila correspondingto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from about 1.10² to about 1.10¹⁵ cfu/g of thecomposition, preferably from about 1.10⁴ to about 1.10¹² cfu/g of thecomposition, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu/g ofthe composition and even more preferably from about 1.10⁶ to about 1.10⁹cfu/g of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila correspondingto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from about 1.10² to about 1.10¹⁵ cfu/mL of thecomposition, preferably from about 1.10⁴ to about 1.10¹² cfu/mL of thecomposition, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu/mL ofthe composition and even more preferably from about 1.10⁶ to about 1.10⁹cfu/mL of the composition.

In another embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila correspondingto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from about 1.10⁶ to about 1.10¹⁰ cfu/g or cfu/mLof the composition, preferably from about 1.10⁸ to about 1.10¹⁰ cfu/g orcfu/mL, more preferably from about 1.10⁹ to about 1.10¹⁰ cfu/g orcfu/mL.

In another embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila correspondingto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from about 1.10⁶ to about 1.10¹¹ cfu/g or cfu/mLof the composition, preferably from about 1.10⁸ to about 1.10¹¹ cfu/g orcfu/mL, more preferably from about 1.10¹⁰ to about 1.10¹¹ cfu/g orcfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila ranging fromabout 1.10² to about 1.10¹⁵ cells/g of the composition, preferably fromabout 1.10⁴ to about 1.10¹² cells/g of the composition, more preferablyfrom about 1.10⁵ to about 1.10¹⁰ cells/g of the composition and evenmore preferably from about 1.10⁶ to about 1.10⁹ cells/g of thecomposition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila ranging fromabout 1.10² to about 1.10¹⁵ cells/mL of the composition, preferably fromabout 1.10⁴ to about 1.10¹² cells/mL of the composition, more preferablyfrom about 1.10⁵ to about 1.10¹⁰ cells/mL of the composition and evenmore preferably from about 1.10⁶ to about 1.10⁹ cells/mL of thecomposition.

In another embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila ranging fromabout 1.10⁶ to about 1.10¹⁰ cells/g or cells/mL of the composition,preferably from about 1.10⁸ to about 1.10¹⁰ cells/g or cells/mL, morepreferably from about 1.10⁹ to about 1.10¹⁰ cells/g or cells/mL.

In another embodiment of the invention, the composition of the inventioncomprises an amount of pasteurized Akkermansia muciniphila ranging fromabout 1.10⁶ to about 1.10¹¹ cells/g or cells/mL of the composition,preferably from about 1.10⁸ to about 1.10¹¹ cells/g or cells/mL, morepreferably from about 1.10¹⁰ to about 1.10¹¹ cells/g or cells/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilacorresponding to an amount of Akkermansia muciniphila before the step ofpasteurization ranging from fragments derived from about 1.10² to about1.10¹⁵ cfu/g or cfu/mL of the composition, preferably from about 1.10⁴to about 1.10¹² cfu/g or cfu/mL of the composition, more preferably fromabout 1.10⁵ to about 1.10¹⁰ cfu/g or cfu/mL of the composition and evenmore preferably from about 1.10⁶ to about 1.10⁹ cfu/g or cfu/mL of thecomposition.

In another embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilacorresponding to an amount of Akkermansia muciniphila before the step ofpasteurization ranging from fragments derived from about 1.10⁶ to about1.10¹⁰ cfu/g or cfu/mL of the composition, preferably from about 1.10⁸to about 1.10¹⁰ cfu/g or cfu/mL, more preferably from about 1.10⁹ toabout 1.10¹⁰ cfu/g or cfu/mL.

In another embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilacorresponding to an amount of Akkermansia muciniphila before the step ofpasteurization ranging from fragments derived from about 1.10⁶ to about1.10¹¹ cfu/g or cfu/mL of the composition, preferably from about 1.10⁸to about 1.10¹¹ cfu/g or cfu/mL, more preferably from about 1.10¹⁰ toabout 1.10¹¹ cfu/g or cfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilaranging from fragments derived from about 1.10² to about 1.10¹⁵ cells/gor cells/mL of the composition, preferably from about 1.10⁴ to about1.10¹² cells/g or cells/mL of the composition, more preferably fromabout 1.10⁵ to about 1.10¹⁰ cells/g or cells/mL of the composition andeven more preferably from about 1.10⁶ to about 1.10⁹ cells/g or cells/mLof the composition.

In another embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilaranging from fragments derived from about 1.10⁶ to about 1.10¹⁰ cells/gor cells/mL of the composition, preferably from about 1.10⁸ to about1.10¹⁰ cells/g or cells/mL, more preferably from about 1.10⁹ to about1.10¹⁰ cells/g or cells/mL.

In another embodiment of the invention, the composition of the inventioncomprises an amount of fragments of pasteurized Akkermansia muciniphilaranging from fragments derived from about 1.10⁶ to about 1.10¹¹ cells/gor cells/mL of the composition, preferably from about 1.10⁸ to about1.10¹¹ cells/g or cells/mL, more preferably from about 1.10¹⁰ to about1.10¹¹ cells/g or cells/mL.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of pasteurized Akkermansia muciniphila ora fragment thereof and at least one pharmaceutically acceptableexcipient. In one embodiment of the invention, the pharmaceuticalcomposition of the invention is for treating or preventing a metabolicdisorder. In another embodiment of the invention, the pharmaceuticalcomposition is for restoring a normal proportion of Akkermansiamuciniphila or increasing the abundance of any active compounds ofAkkermansia muciniphila in the gut of a subject in need thereof.

As used herein the term “pharmaceutically acceptable excipient” refersto an excipient that does not produce an adverse, allergic or otheruntoward reaction when administered to an animal, preferably a human. Itmay include any and all solvents, dispersion media, coatings, isotonicand absorption delaying agents and the like. For human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biologics standards.

The present invention also relates to a medicament comprising aneffective amount of pasteurized Akkermansia muciniphila or a fragmentthereof. In one embodiment of the invention, the medicament of theinvention is for treating or preventing a metabolic disorder. In anotherembodiment of the invention, the medicament is for restoring a normalproportion of Akkermansia muciniphila in the gut of a subject in needthereof.

The present invention also relates to a method for treating orpreventing a metabolic disorder in a subject in need thereof, whereinsaid method comprises administering an effective amount of pasteurizedAkkermansia muciniphila or a fragment thereof to the subject.

Another object of the invention is a method for restoring a normalproportion of Akkermansia muciniphila, fragments or other activecompounds of Akkermansia muciniphila in the gut of a subject in needthereof, wherein said method comprises administering an effective amountof pasteurized Akkermansia muciniphila or a fragment thereof to thesubject.

In one embodiment, the method of the invention comprises administeringan effective amount of the composition, of the pharmaceuticalcomposition or of the medicament of the invention to the subject.

In one embodiment of the invention, pasteurized Akkermansia muciniphilaor a fragment thereof, or the composition, pharmaceutical composition ormedicament is administered at least once a week, preferably at leasttwice a week, more preferably at least three times a week, and even morepreferably at least four times a week. In another embodiment,pasteurized Akkermansia muciniphila or a fragment thereof, or thecomposition, pharmaceutical composition or medicament is administered atleast once a day, and preferably at least twice a day.

In one embodiment, pasteurized Akkermansia muciniphila or a fragmentthereof, or the composition, pharmaceutical composition or medicament ofthe invention is administered during 1 week, preferably during 2, 3, 4,5, 6, 7 or 8 weeks or more.

In one embodiment, pasteurized Akkermansia muciniphila or a fragmentthereof, or the composition, pharmaceutical composition or medicament ofthe invention is administered for a period that lasts until the desiredoutcome is achieved (e.g., weight loss, metabolic disorder treatment,decrease of cholesterol plasma level . . . ).

In one embodiment, the administration of pasteurized Akkermansiamuciniphila or a fragment thereof, or the composition, pharmaceuticalcomposition or medicament of the invention is permanent, i.e. is notlimited in time.

In one embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10² to about 1.10¹⁵ cfu/day, preferably from about 1.10⁴ to about1.10¹² cfu/day, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu/dayand even more preferably from about 1.10⁶ to about 1.10⁹ cfu/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10⁶ to about 1.10¹⁰ cfu/day, preferably from about 1.10⁸ to about1.10¹⁰ cfu/day, more preferably from about 1.10⁹ to about 1.10¹⁰cfu/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10⁶ to about 1.10¹¹ cfu/day, preferably from about 1.10⁸ to about1.10¹¹ cfu/day, more preferably from about 1.10¹⁰ to about 1.10¹¹cfu/day.

In one embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10² to about1.10¹⁵ cells/day, preferably from about 1.10⁴ to about 1.10¹² cells/day,more preferably from about 1.10⁵ to about 1.10¹⁰ cells/day and even morepreferably from about 1.10⁶ to about 1.10⁹ cells/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10⁶ to about1.10¹⁰ cells/day, preferably from about 1.10⁸ to about 1.10¹⁰ cells/day,more preferably from about 1.10⁹ to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10⁶ to about1.10¹¹ cells/day, preferably from about 1.10⁸ to about 1.10¹¹ cells/day,more preferably from about 1.10¹⁰ to about 1.10¹¹ cells/day.

In one embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from 1.10² to about 1.10¹⁵ cfu/day,preferably from about 1.10⁴ to about 1.10¹² cfu/day, more preferablyfrom about 1.10⁵ to about 1.10¹⁰ cfu/day and even more preferably fromabout 1.10⁶ to about 1.10⁹ cfu/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from 1.10⁶ to about 1.10′⁰ cfu/day,preferably from about 1.10⁸ to about 1.10¹⁰ cfu/day, more preferablyfrom about 1.10⁹ to about 1.10¹⁰ cfu/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from 1.10⁶ to about 1.10¹¹ cfu/day,preferably from about 1.10⁸ to about 1.10¹¹ cfu/day, more preferablyfrom about 1.10¹⁰ to about 1.10¹¹ cfu/day.

In one embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from 1.10² to about 1.10¹⁵ cells/day, preferably fromabout 1.10⁴ to about 1.10¹² cells/day, more preferably from about 1.10⁵to about 1.10¹⁰ cells/day and even more preferably from about 1.10⁶ toabout 1.10⁹ cells/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from 1.10⁶ to about 1.10¹⁰ cells/day, preferably fromabout 1.10⁸ to about 1.10¹⁰ cells/day, more preferably from about 1.10⁹to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from 1.10⁶ to about 1.10¹¹ cells/day, preferably fromabout 1.10⁸ to about 1.10¹¹ cells/day, more preferably from about 1.10¹⁰to about 1.10¹¹ cells/day.

In one embodiment of the invention, the subject is overweight. Inanother embodiment, the subject is obese.

In one embodiment of the invention, the subject is diagnosed with ametabolic disorder, such as, for example, with an overweight and/orobesity related metabolic disorder. In one embodiment of the invention,the subject is diagnosed with a metabolic disorder, such as, forexample, with a normal weight and/or impaired fasting glucose and/orhypertriglyceridemia and/or any related metabolic disorder orcardiovascular risk factor.

In another embodiment, the subject is at risk of developing a metabolicdisorder, such as, for example, an overweight and/or obesity relatedmetabolic disorder. In one embodiment, said risk is related to the factthat the subject is overweight or obese. In another embodiment, saidrisk corresponds to a predisposition, such as, for example, a familialpredisposition to a metabolic disorder, such as, for example, to anoverweight and/or obesity related metabolic disorder.

In one embodiment of the invention, the subject presents a deregulationof the gut microbiota composition. Preferably, the gut microbiota ofsaid subject is depleted in Akkermansia muciniphila strains. In oneembodiment, the proportion of Akkermansia muciniphila in the gut of thesubject is inferior to 1%, preferably inferior to 0.5%, more preferablyinferior to 0.1%, in number of Akkermansia muciniphila cells to thetotal number of bacterial cells in the gut.

The present invention also relates to the cosmetic use of pasteurizedAkkermansia muciniphila or a fragment thereof for promoting weight lossin a subject.

Another object of the invention is thus a cosmetic compositioncomprising a cosmetically effective amount of pasteurized Akkermansiamuciniphila or a fragment thereof, and the use thereof for promotingweight loss in a subject. As used herein, a “cosmetically effectiveamount” refers to the amount of a cosmetic composition necessary andsufficient for promoting a cosmetic effect, such as, for example, forinducing weight loss in a subject.

The present invention also relates to a method for promoting weight lossin a subject in need thereof, wherein said method comprisesadministering a cosmetically effective amount of pasteurized Akkermansiamuciniphila or a fragment thereof to said subject.

In one embodiment, the method of the invention comprises administering acosmetically effective amount of the composition or of the cosmeticcomposition of the invention to the subject.

In one embodiment of the invention, the cosmetically effective amount ofpasteurized Akkermansia muciniphila corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging fromabout 1.10² to about 1.10¹⁵ cfu, preferably from about 1.10⁴ to about1.10¹² cfu, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu andeven more preferably from about 1.10⁶ to about 1.10⁹ cfu.

In another embodiment of the invention, the cosmetically effectiveamount of pasteurized Akkermansia muciniphila corresponds to an amountof Akkermansia muciniphila before the step of pasteurization rangingfrom about 1.10⁶ to about 1.10¹⁰ cfu, preferably from about 1.10⁸ toabout 1.10¹⁰ cfu, more preferably from about 1.10⁹ to about 1.10¹⁰ cfu.

In another embodiment of the invention, the cosmetically effectiveamount of pasteurized Akkermansia muciniphila corresponds to an amountof Akkermansia muciniphila before the step of pasteurization rangingfrom about 1.10⁶ to about 1.10¹¹ cfu, preferably from about 1.10⁸ toabout 1.10¹¹ cfu, more preferably from about 1.10¹⁰ to about 1.10¹¹ cfu.

In one embodiment of the invention, the cosmetically effective amount ofpasteurized Akkermansia muciniphila ranges from about 1.10² to about1.10¹⁵ cells, preferably from about 1.10⁴ to about 1.10¹² cells, morepreferably from about 1.10⁵ to about 1.10¹⁰ cells and even morepreferably from about 1.10⁶ to about 1.10⁹ cells.

In another embodiment of the invention, the cosmetically effectiveamount of pasteurized Akkermansia muciniphila ranges from about 1.10⁶ toabout 1.10¹⁰ cells, preferably from about 1.10⁸ to about 1.10¹⁰ cells,more preferably from about 1.10⁹ to about 1.10¹⁰ cells.

In another embodiment of the invention, the cosmetically effectiveamount of pasteurized Akkermansia muciniphila ranges from about 1.10⁶ toabout 1.10¹¹ cells, preferably from about 1.10⁸ to about 1.10¹¹ cells,more preferably from about 1.10¹⁰ to about 1.10¹¹ cells.

In one embodiment of the invention, the cosmetically effective amount offragments of pasteurized Akkermansia muciniphila corresponds to anamount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from about 1.10² to about 1.10¹′ cfu,preferably from about 1.10⁴ to about 1.10¹² cfu, more preferably fromabout 1.10⁵ to about 1.10¹⁰ cfu and even more preferably from about1.10⁶ to about 1.10⁹ cfu.

In another embodiment of the invention, the cosmetically effectiveamount of fragments of pasteurized Akkermansia muciniphila correspondsto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from fragments derived from about 1.10⁶ to about1.10¹⁰ cfu, preferably from about 1.10⁸ to about 1.10¹⁰ cfu, morepreferably from about 1.10⁹ to about 1.10¹⁰ cfu.

In another embodiment of the invention, the cosmetically effectiveamount of fragments of pasteurized Akkermansia muciniphila correspondsto an amount of Akkermansia muciniphila before the step ofpasteurization ranging from fragments derived from about 1.10⁶ to about1.10¹¹ cfu, preferably from about 1.10⁸ to about 1.10¹¹ cfu, morepreferably from about 1.10¹⁰ to about 1.10¹¹ cfu.

In one embodiment of the invention, the cosmetically effective amount offragments of pasteurized Akkermansia muciniphila ranges from fragmentsderived from about 1.10² to about 1.10¹⁵ cells, preferably from about1.10⁴ to about 1.10¹² cells, more preferably from about 1.10⁵ to about1.10¹⁰ cells and even more preferably from about 1.10⁶ to about 1.10⁹cells.

In another embodiment of the invention, the cosmetically effectiveamount of fragments of pasteurized Akkermansia muciniphila ranges fromfragments derived from about 1.10⁶ to about 1.10¹⁰ cells, preferablyfrom about 1.10⁸ to about 1.10¹⁰ cells, more preferably from about 1.10⁹to about 1.10¹⁰ cells.

In another embodiment of the invention, the cosmetically effectiveamount of fragments of pasteurized Akkermansia muciniphila ranges fromfragments derived from about 1.10⁶ to about 1.10¹¹ cells, preferablyfrom about 1.10⁸ to about 1.10¹¹ cells, more preferably from about1.10¹⁰ to about 1.10¹¹ cells.

In one embodiment of the invention, pasteurized Akkermansia muciniphilaor a fragment thereof, or the composition or cosmetic composition isadministered at least once a week, preferably at least twice a week,more preferably at least three times a week, and even more preferably atleast four times a week. In another embodiment, pasteurized Akkermansiamuciniphila or a fragment thereof, or the composition or cosmeticcomposition is administered at least once a day, and preferably at leasttwice a day.

In one embodiment, pasteurized Akkermansia muciniphila or a fragmentthereof, or the composition or cosmetic composition of the invention isadministered during 1 week, preferably 2, 3, 4, 5, 6, 7 or 8 weeks ormore.

In one embodiment, pasteurized Akkermansia muciniphila or a fragmentthereof, or the composition or cosmetic composition of the invention isadministered for a period that lasts until the desired outcome isachieved (e.g., weight loss . . . ).

In one embodiment, the administration of pasteurized Akkermansiamuciniphila or a fragment thereof, or the composition or cosmeticcomposition of the invention is permanent, i.e. is not limited in time.

In one embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10² to about 1.10¹⁵ cfu/day, preferably from about 1.10⁵ to about1.10¹² cfu/day, more preferably from about 1.10⁸ to about 1.10¹⁰cfu/day, and even more preferably from about 1.10⁹ to about 1.10¹⁰cfu/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10⁶ to about 1.10¹⁰ cfu/day, preferably from about 1.10⁸ to about1.10¹⁰ cfu/day, more preferably from about 1.10⁹ to about 1.10¹⁰cfu/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day corresponds to an amount ofAkkermansia muciniphila before the step of pasteurization ranging from1.10⁶ to about 1.10¹¹ cfu/day, preferably from about 1.10⁸ to about1.10¹¹ cfu/day, more preferably from about 1.10¹⁰ to about 1.10¹¹cfu/day.

In one embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10² to about1.10¹⁵ cells/day, preferably from about 1.10⁵ to about 1.10¹² cells/day,more preferably from about 1.10⁸ to about 1.10¹⁰ cells/day, and evenmore preferably from about 1.10⁹ to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10⁶ to about1.10¹⁰ cells/day, preferably from about 1.10⁸ to about 1.10¹⁰ cells/day,more preferably from about 1.10⁹ to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of pasteurizedAkkermansia muciniphila administered per day ranges from 1.10⁶ to about1.10¹¹ cells/day, preferably from about 1.10⁸ to about 1.10¹¹ cells/day,more preferably from about 1.10¹⁰ to about 1.10¹¹ cells/day.

In one embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from about 1.10² to about 1.10¹⁵ cfu/day,preferably from about 1.10⁵ to about 1.10¹² cfu/day, more preferablyfrom about 1.10⁸ to about 1.10¹⁰ cfu/day, and even more preferably fromabout 1.10⁹ to about 1.10¹⁰ cfu/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from about 1.10⁶ to about 1.10¹⁰ cfu/day,preferably from about 1.10⁸ to about 1.10¹⁰ cfu/day, more preferablyfrom about 1.10⁹ to about 1.10¹⁰ cfu/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day corresponds toan amount of Akkermansia muciniphila before the step of pasteurizationranging from fragments derived from about 1.10⁶ to about 1.10¹¹ cfu/day,preferably from about 1.10⁸ to about 1.10¹¹ cfu/day, more preferablyfrom about 1.10¹⁰ to about 1.10¹¹ cfu/day.

In one embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from about 1.10² to about 1.10¹⁵ cells/day, preferablyfrom about 1.10⁵ to about 1.10¹² cells/day, more preferably from about1.10⁸ to about 1.10¹⁰ cells/day, and even more preferably from about1.10⁹ to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from about 1.10⁶ to about 1.10¹⁰ cells/day, preferablyfrom about 1.10⁸ to about 1.10¹⁰ cells/day, more preferably from about1.10⁹ to about 1.10¹⁰ cells/day.

In another embodiment of the invention, the daily amount of fragments ofpasteurized Akkermansia muciniphila administered per day ranges fromfragments derived from about 1.10⁶ to about 1.10¹¹ cells/day, preferablyfrom about 1.10⁸ to about 1.10¹¹ cells/day, more preferably from about1.10¹⁰ to about 1.10¹¹ cells/day.

In one embodiment, said subject is not an obese subject. In anotherembodiment, said subject is overweight.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament furthercomprises additional probiotic strains or species, such as, for example,bacterial probiotic strains or species; prokaryotes probiotics otherthan bacteria; or fungal strains or species, preferably yeast strains orspecies. In one embodiment, said additional probiotic strains or speciesare selected from those naturally present in the gut of the subject,preferably in the human gut, more preferably in the gut of healthy humansubjects.

Examples of bacterial probiotic strains or species that may be used inthe present invention include, but are not limited to Lactobacillus,Laciococcus, Bifidobacterium, Veillonella, Desemzia, Christensenella,Allobaculum, Coprococcus, Collinsella, Citrobacter, Turicibacter,Sutterella, Subdoligranulum, Streptococcus, Sporobacter,Sporacetigenium, Ruminococcus, Roseburia, Proteus, Propionobacterium,Leuconostoc, Weissella, Pediococcus, Streptococcus, Prevotella,Parabacteroides, Papillibacter, Oscillospira, Melissococcus, Dorea,Dialister, Clostridium, Cedecea, Catenibacterium, Butyrivibrio,Buttiauxella, Bulleidia, Bilophila, Bacteroides, Anaerovorax,Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes, Atopobium,Alistipes, Acinetobacter, Slackie, Shigella, Shewanella, Serratia,Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium,Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella.

In one particular embodiment, said bacterial probiotic strains orspecies are selected from the list comprising Bifidobacterium andLactobacillus. In one embodiment, Bifidobacterium probiotic strains orspecies are preferably selected from the group comprisingBifidobacterium animalis, more preferably Bifidobacterium animalis spp.lactis, and Bifidobacterium lactis. In one embodiment, Lactobacillusprobiotic strains or species are preferably selected from the groupcomprising Lactobacillus rhamnosus, Lactobacillus casei andLactobacillus acidophilus.

Examples of prokaryote strains or species that may be used in thepresent invention include, but are not limited to Archaea, Firmicutes,Verrucomicrobia, Christensenella, Bacteroidetes (such as, for example,Allistipes, Bacteroides ovatus, Bacteroides splachnicus, Bacteroidesstercoris, Parabacteroides, Prevotella ruminicola, Porphyromondaceae,and related genus), Proteobacteria, Betaproteobacteria (such as, forexample, Aquabacterium and Burkholderia), Gammaproteobacteria (such as,for example, Xanthomonadaceae), Actinobacteria (such as, for example,Actinomycetaceae and Atopobium), Fusobacteria, Methanobacteria,Spirochaetes, Fibrobacteres, Deferribacteres, Deinococcus, Thermus,Cyanobacteria, Methanobrevibacteria, Peptostreptococcus, Ruminococcus,Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella,Roseburia, Dialister, Anaerotruncus, Staphylococcus, Micrococcus,Propionobacteria, Enterobacteriaceae, Faecalibacterium, Bacteroides,Parabacteroides, Prevotella, Eubacterium, Bacilli (such as, for example,Lactobacillus salivarius and related species, Aerococcus,Granulicatella, Streptococcus bovis and related genus and Streptococcusintermedius and related genus), Clostridium (such as, for example,Eubacterium hallii, Eubacterium limosum and related genus) andButyrivibrio.

Examples of fungal probiotic strains or species, preferably yeastprobiotic strains or species that may be used in the present inventioninclude, but are not limited Ascomycetes, Zygomycetes andDeuteromycetes, preferably from the groups Aspergillus, Torulopsis,Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora,Bretanomyces, Pichia, Amylomyces, Zygosaccharomyces, Endomycess,Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus,Yarrowia, Endomyces, Debaryomyces, and/or Penicillium.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament does notcomprise the bacterial strains Lactobacillus-Enterococcus, Bacteroidesand/or Atopobium.

In one embodiment of the invention, the only one microbial strain orspecies, preferably bacterial strain or species, comprised in thecomposition, pharmaceutical composition, cosmetic composition ormedicament is Akkermansia muciniphila.

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament consists of pasteurizedAkkermansia muciniphila.

In another embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament consists essentially ofpasteurized Akkermansia muciniphila, wherein “consisting essentially of”herein means that Akkermansia muciniphila is the only microbial strainor species, preferably the only bacterial strain or species comprised inthe composition, pharmaceutical composition, cosmetic composition ormedicament.

In one embodiment of the invention, pasteurized Akkermansia muciniphilaor a fragment thereof activates or inhibits the growth and/or biologicalactivity of other bacterial strain(s) or species of the gut microbiota.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament furthercomprises a prebiotic.

Examples of prebiotics that may be used in the present inventioninclude, but are not limited to, inulin and inulin-type fructans,oligofructose, beta-glucans, xylose, arabinose, arabinoxylan, ribose,galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose,glucose, esculin, tween 80, trehalose, maltose, mannose, mellibiose,mucus or mucins, raffinose, fructooligosaccharides,galacto-oligosaccharides, amino acids, alcohols, fermentablecarbohydrates and any combinations thereof.

Other non-limiting examples of prebiotics include water-solublecellulose derivatives, water-insoluble cellulose derivatives,unprocessed oatmeal, metamucil, all-bran, and any combinations thereof.

Examples of water-soluble cellulose derivatives include, but are notlimited to, methylcellulose, methyl ethyl cellulose, hydroxyethylcellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethylcellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose,hydroxypropyl methylcellulose, and carboxymethyl cellulose.

Pasteurized Akkermansia muciniphila or a fragment thereof or thecomposition, pharmaceutical composition, cosmetic composition ormedicament of the invention may be administered by several routes ofadministration. Examples of adapted routes of administration include,but are not limited to, oral administration, rectal administration,administration via esophagogastroduodenoscopy, administration viacolonoscopy, administration using a nasogastric or orogastric tube andthe like.

According to an embodiment, pasteurized Akkermansia muciniphila or afragment thereof or the composition, pharmaceutical composition,cosmetic composition or medicament of the invention is in a form adaptedto oral administration. According to a first embodiment, the formadapted to oral administration is a solid form selected from the groupcomprising tablets, pills, capsules, soft gelatin capsules, sugarcoatedpills, orodispersing tablets, effervescent tablets or other solids.According to a second embodiment, the form adapted to oraladministration is a liquid form, such as, for example, a drinkablesolution, liposomal forms and the like.

In one embodiment, the composition, pharmaceutical composition, cosmeticcomposition or medicament of the invention further comprises excipients,diluent and/or carriers selected with regard to the intended route ofadministration. Examples of excipients, diluent and/or carriers include,but are not limited to, water, phosphate buffer saline, anaerobicphosphate buffer saline, sodium bicarbonate, juice, milk, yogurt, infantformula, dairy product, coloring agents, such as, for example, titanedioxide (E171), iron dioxide (E172) and brilliant black BN (E151);flavoring agents; thickeners, such as, for example, glycerolmonostearate; sweeteners; coating agents, such as, for example, refinedcolza oil, soya oil, peanut oil, soya lecithin or fish gelatin; dilutingagents, such as, for example, lactose, monohydrated lactose or starch;binding agents, such as, for example, povidone, pregelatinized starch,gums, saccharose, polyethylene glycol (PEG) 4000 or PEG 6000;disintegrating agents, such as, for example, microcrystalline celluloseor sodium carboxymethyl starch, such as, for example, sodiumcarboxymethyl starch type A; lubricant agents, such as, for example,magnesium stearate; flow agent, such as, for example, colloidalanhydrous silica, etc. . . . .

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament is in the form of anutritional composition, i.e. comprises liquid or solid food, feed ordrinking water. In one embodiment of the invention, the composition,pharmaceutical composition, cosmetic composition or medicament is a foodproduct, such as, for example, dairy products, dairy drinks, yogurt,fruit or vegetable juice or concentrate thereof, powders, malt or soy orcereal based beverages, breakfast cereal such as muesli flakes, fruitand vegetable juice powders, cereal and/or chocolate bars,confectionary, spreads, flours, milk, smoothies, confectionary, milkproduct, milk powder, reconstituted milk, cultured milk, yoghurt,drinking yoghurt, set yoghurt, drink, dairy drink, milk drink,chocolate, gels, ice creams, cereals, reconstituted fruit products,snack bars, food bars, muesli bars, spreads, sauces, dips, dairyproducts including yoghurts and cheeses, drinks including dairy andnon-dairy based drinks, sports supplements including dairy and non-dairybased sports supplements.

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament is in the form of a foodadditive, drink additive, dietary supplement, nutritional product,medical food or nutraceutical composition.

It is known that obesity and related disorders are associated with anincreased gut permeability and with impaired mucus production,epithelium barrier, immune system and/or antibacterial compoundsproduction by the subject; and the Applicant suggests that theadministration of pasteurized Akkermansia muciniphila may restore theseparameters. Therefore, the present invention also relates to pasteurizedAkkermansia muciniphila or a fragment thereof for decreasing gutpermeability and/or for restoring impaired mucus production and/or forrestoring epithelium barrier and/or for restoring immune system and/orfor restoring the production of antibacterial compounds. Another objectof the invention is a method for decreasing gut permeability and/or forrestoring impaired mucus production and/or for restoring epitheliumbarrier and/or for restoring immune system and/or for restoring theproduction of antibacterial compounds in a subject in need thereof,comprising administering an effective or cosmetically effective amountof pasteurized Akkermansia muciniphila or a fragment thereof to asubject in need thereof Therefore, the present invention also relates topasteurized Akkermansia muciniphila or a fragment thereof forcontrolling gut barrier function, and to a method for controlling gutbarrier function comprising administering an effective or cosmeticallyeffective amount of pasteurized Akkermansia muciniphila or a fragmentthereof to a subject in need thereof. In one embodiment, pasteurizedAkkermansia muciniphila or a fragment thereof regulates mucus layerthickness (which may be decreased in obesity or other metabolicdisorders).

In another embodiment, the administration of pasteurized Akkermansiamuciniphila or a fragment thereof induces the production of colonantimicrobial peptides, such as, for example, RegIIIgamma. In anotherembodiment, the administration of pasteurized Akkermansia muciniphila ora fragment thereof induces the production of compounds of theendocannabinoids family, such as, for example, acylglycerols selectedfrom the group comprising 2-oleoylglycerol, 2-palmitoylglycerol and2-arachidonoylglycerol. In another embodiment, the administration ofpasteurized Akkermansia muciniphila or a fragment thereof regulatesmucus turnover.

Another object of the invention concerns pasteurized Akkermansiamuciniphila or a fragment thereof for use in treating metabolicdysfunction associated with or caused by a metabolic disorder. Stillanother object of the invention is thus a method for treating metabolicdysfunction associated with or caused by a metabolic disorder in asubject in need thereof, comprising administering an effective amount ora cosmetically effective amount of pasteurized Akkermansia muciniphilaor a fragment thereof to a subject in need thereo.

The Applicant also showed that the administration of pasteurizedAkkermansia muciniphila controls fat storage and adipose tissuemetabolism. Therefore, another object of the invention concernspasteurized Akkermansia muciniphila or a fragment thereof for use incontrolling fat storage and adipose tissue metabolism. Another object ofthe invention is also a method for controlling fat storage and adiposetissue metabolism comprising administering an effective amount or acosmetically effective amount of pasteurized Akkermansia muciniphila ora fragment thereof to a subject in need thereof. In one embodiment, saidcontrol does not involve any change in food intake. In one embodiment ofthe invention, administration of pasteurized Akkermansia muciniphila ora fragment thereof abolishes metabolic endotoxemia. In anotherembodiment, administration of pasteurized Akkermansia muciniphila or afragment thereof lowers fat mass. In another embodiment, administrationof pasteurized Akkermansia muciniphila or a fragment thereof increasesmRNA expression of markers of adipocyte differentiation and lipidoxidation, preferably without affecting lipogenesis.

The present invention also relates to pasteurized Akkermansiamuciniphila or a fragment thereof for use in the regulation of adiposetissue metabolism and glucose homeostasis; and to a method forregulating adipose tissue metabolism and glucose homeostasis comprisingadministering an effective amount or a cosmetically effective amount ofpasteurized Akkermansia muciniphila or a fragment thereof to a subjectin need thereof. In one embodiment of the invention, the administrationof pasteurized Akkermansia muciniphila or a fragment thereof reversesdiet-induced fasting hyperglycemia. In another embodiment, theadministration of pasteurized Akkermansia muciniphila or a fragmentthereof induces a reduction of at least 10%, preferably of at least 30%,more preferably of at least 40% of hepatic glucose-6-phosphataseexpression. In another embodiment, the administration of pasteurizedAkkermansia muciniphila or a fragment thereof induces a reduction of theinsulin-resistance index. In one embodiment, said reduction of theinsulin-resistance index is of at least 5%, preferably of at least 10%,more preferably of at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

The Applicant showed that the administration of pasteurized Akkermansiamuciniphila decreases glucose intolerance and insulin resistance inhigh-fat diet fed mice. Therefore, the present invention also relates topasteurized Akkermansia muciniphila or a fragment thereof for decreasingglucose intolerance and/or insulin resistance; and to a method fordecreasing glucose intolerance and/or insulin resistance comprisingadministering an effective amount or a cosmetically effective amount ofpasteurized Akkermansia muciniphila or a fragment thereof to a subjectin need thereof.

The present invention also relates to pasteurized Akkermansiamuciniphila or a fragment thereof for treating inflammation, preferablylow grade inflammation, associated with or caused by metabolicdisorders; and to a method for treating inflammation related tometabolic disorders comprising administering an effective amount or acosmetically effective amount of pasteurized Akkermansia muciniphila ora fragment thereof to a subject in need thereof.

The Applicant showed that the administration of pasteurized Akkermansiamuciniphila decreases plasma triglycerides levels in treated mice.Therefore, the present invention also relates to pasteurized Akkermansiamuciniphila or a fragment thereof for decreasing plasma triglycerideslevels; and to a method for decreasing plasma triglycerides levelscomprising administering an effective amount or a cosmetically effectiveamount of pasteurized Akkermansia muciniphila or a fragment thereof to asubject in need thereof.

The present invention also relates to pasteurized Akkermansiamuciniphila or a fragment thereof for decreasing plasma cholesterol; andto a method for decreasing plasma cholesterol comprising administeringan effective amount or a cosmetically effective amount of pasteurizedAkkermansia muciniphila or a fragment thereof to a subject in needthereof.

In one embodiment of the invention, the administration of pasteurizedAkkermansia muciniphila or a fragment thereof to a subject has no impacton food intake of said subject.

In one embodiment of the invention, the administration of pasteurizedAkkermansia muciniphila or a fragment thereof to a subject increasesenergy expenditure of said subject, preferably without impacting thefood intake of said subject.

The present invention thus also relates to a method of increasing energyexpenditure of a subject, comprising administering pasteurizedAkkermansia muciniphila or a fragment thereof, or a composition,pharmaceutical composition, cosmetic composition or medicament of theinvention to the subject, preferably in a therapeutically orcosmetically effective amount. Preferably, the method of the inventiondoes not comprise or further comprise modulating the food intake of saidsubject. In one embodiment of the invention, the method of the inventionincreases energy expenditure, thereby inducing durable weight loss inthe subject, and thereby treating metabolic disorders in said subject,such as, for example, obesity related metabolic disorders.

In one embodiment, the administration of pasteurized Akkermansiamuciniphila or a fragment thereof to a subject increases satiety in saidsubject. Consequently, according to this embodiment, the method of theinvention increases satiety in a subject, thereby inducing durableweight loss in the subject, and thereby treating metabolic disorders insaid subject, such as, for example, obesity related metabolic disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of histograms showing that Akkermansia muciniphila grownon a mucus-based medium or on a non-mucus-based growth mediumcounteracts the increase in body weight gain and fat mass gain in micefed a high-fat diet. Furthermore, effects of pasteurized A. muciniphilaon body weight gain and fat mass gain are stronger than with the livebacterium. (a) Total body weight gain (g) in mice fed a control diet (CTND), a high-fat diet (CT HFD) and treated daily by oral gavage withsterile anaerobic PBS containing glycerol or mice fed a high-fat dietand treated daily by oral gavage with live A. muciniphila grown on amucus-based medium (HFD Akk M), a non-mucus-based medium (HFD Akk G), orA. muciniphila grown on a mucus-based medium and pasteurized (HFD Akk P)for 4 weeks (n=8-10). (2.10⁸ bacterial cells suspended in 150 μL ofsterile anaerobic PBS). (b) Total fat mass gain (g) measured bytime-domain nuclear magnetic resonance (n=8-10). Data are shown asmean±SEM. Data with different superscript letters are significantlydifferent (P<0.05), according to one-way ANOVA analysis followed byTukey post-hoc test.

FIG. 2 is a histogram showing normalization of the adiposity index ofhigh-fat diet-fed mice after treatment with A. muciniphila. Adiposityindex (g) shown as the addition of the weight of the epididymal,subcutaneous and mesenteric adipose depots (n=8-10). Data are shown asmean±SEM. * corresponds to P value <0.05 when two conditions werecompared with an unpaired two-tailed Student's t-test.

FIG. 3 is a set of graphs showing a diminution of glucose intolerance inmice fed a high-fat diet after administration of pasteurized A.muciniphila to a higher extent than administration of live A.muciniphila grown either on a mucus-based or non-mucus-based medium. (a)Plasma glucose profile following 2 g/kg glucose oral challenge in freelymoving mice (n=8-10). (b) Mean area under the curve (AUC) measuredbetween −30 and 120 min after glucose load (n=8-10). (c) Insulinresistance index, determined by multiplying the AUC of plasma glucose(−30 to 120 min) by the AUC of plasma insulin (−30 to 15 min) (n=8-10).Data are shown as mean±SEM. Data with different superscript letters aresignificantly different (P<0.05), according to one-way ANOVA analysisfollowed by Tukey post-hoc test.

FIG. 4 is a histogram showing modulation of the expression of markers ofintestinal integrity and corrects HFD-induced metabolic endotoxemiaafter administration of A. muciniphila grown on a non-mucus-based mediumand either live or pasteurized. (a) mRNA expression of Ocln, Cldn3 andLyz1 in the jejunum (n=7-10), (b) mRNA expression of Ocln, Cldn3 andLyz1 in the ileum (n=7-10), (c) Plasma Lipopolysaccharide levels (EU/mL)(n=5-9). Data are shown as mean±SEM. Data with different superscriptletters are significantly different (P<0.05), according to one-way ANOVAanalysis followed by Tukey post-hoc test.

FIG. 5 is a set of histograms showing a reduction of body weight gainand fat mass gain after administration of pasteurized A. muciniphila toa higher extent than live A. muciniphila grown on a non-mucus-basedmedium. (a) Total body weight gain (g) in mice fed a control diet (CTND), a high-fat diet (CT HFD) and treated daily by oral gavage withsterile anaerobic PBS containing glycerol or mice fed a high-fat dietand treated daily by oral gavage with live A. muciniphila grown on anon-mucus-based medium (HFD Akk G), or A. muciniphila grown on amucus-based medium and pasteurized (HFD Akk P) (n=16-19) for 5 weeks.(2.10⁸ bacterial cells suspended in 150 μL of sterile anaerobic PBS).(b) Total fat mass gain (g) measured by time-domain nuclear magneticresonance (n=16-19). Data are shown as mean±SEM. Data with differentsuperscript letters are significantly different (P<0.05), according toone-way ANOVA analysis followed by Tukey post-hoc test.

FIG. 6 is a histogram showing a reduction of adiposity index afteradministration of pasteurized A. muciniphila to a higher extent thanlive A. muciniphila grown on a non-mucus-based medium. Adiposity index(g), shown as the combined weight of the epididymal, subcutaneous andmesenteric adipose depots (n=16-19). Data are shown as mean±SEM. Datawith different superscript letters are significantly different (P<0.05),according to one-way ANOVA analysis followed by Tukey post-hoc test.

FIG. 7 is a set of histograms showing that administration of pasteurizedA. muciniphila counteracts glucose intolerance in mice fed a high-fatdiet to a higher extent than live A. muciniphila. Mice were fed acontrol diet (CT ND), a high-fat diet (CT HFD) and treated daily by oralgavage with sterile anaerobic PBS containing glycerol or A. muciniphilagrown on a non-mucus-based medium, either live (HFD Akk G), orpasteurized (HFD Akk P) for 5 weeks. (a) Plasma glucose profilefollowing 2 g/kg glucose oral challenge in freely moving mice (n=8-10).Data with different superscript letters are significantly different(P<0.05), according to two-way ANOVA analysis followed by Bonferonnipost-test. (b) Mean area under the curve (AUC) measured between −30 and120 min after glucose load (n=10). (c) Insulin resistance index,determined by multiplying the AUC of plasma glucose (−30 to 120 min) bythe AUC of plasma insulin (−30 to 15 min) (n=8-10). Data are shown asmean±SEM. Data with different superscript letters are significantlydifferent (P<0.05), according to one-way ANOVA analysis followed byTukey post-hoc test.

FIG. 8 is a set of graphs showing that administration of either live orpasteurized A. muciniphila counteracts glucose intolerance and insulinresistance in mice fed a high-fat diet. (a) Plasma glucose profilefollowing 2 g/kg glucose oral challenge in freely moving mice (n=8-10).Data are shown as mean±SEM. Data with different superscript letters aresignificantly different (P<0.05), according to two-way ANOVA analysisfollowed by Bonferonni post-test. (b) Mean area under the curve (AUC)measured between −30 and 120 min after glucose load (n=8-10). Data areshown as mean±SEM. Data with different superscript letters aresignificantly different (P<0.05), according to one-way ANOVA analysisfollowed by Tukey post-hoc test. (c) Ratio of the control (−) andinsulin-stimulated (+) p-IRβ on the loading control as measured bydensitometry. (d) Ratio of the control and insulin-stimulatedp-Akt^(thr308) on the loading control as measured by densitometry. (e)Ratio of the control and insulin-stimulated p-Akt^(ser473) on theloading control as measured by densitometry. (c-e) n=3-5. Data withdifferent superscript letters are significantly different (P<0.05),according to two-way ANOVA analysis followed by Bonferonni post-test.

FIG. 9 is a photograph (a) and a histogram (b) showing thatadministration of pasteurized A. muciniphila counteracts the effects ofa high-fat diet on the mean adipocyte diameter. Mice were fed a controldiet (CT ND), a high-fat diet (CT HFD) and treated daily by oral gavagewith sterile anaerobic PBS containing glycerol or A. muciniphila grownon a non-mucus-based medium, either live (HFD Akk G), or pasteurized(HFD Akk P) for 5 weeks. (a) Representative haematoxylin andeosin-stained picture of subcutaneous adipose tissue depots. Scale bar:100 μm. (b) Mean adipocyte diameter (μm) determined by histologicalanalysis (n=16-19). (c) Leptin plasma levels measured in the portal vein(pg/mL) (n=8-10). Data are shown as mean±SEM. Data with differentsuperscript letters are significantly different (P<0.05), according toone-way ANOVA analysis followed by Tukey pose-hoc test.

FIG. 10 is a histogram showing the reduction of plasma triglycerideslevels after administration of pasteurized A. muciniphila. Mice were feda control diet (CT ND), a high-fat diet (CT HFD) and treated daily byoral gavage with sterile anaerobic PBS containing glycerol or A.muciniphila grown on a non-mucus-based medium, either live (HFD Akk G),or pasteurized (HFD Akk P) for 5 weeks (n=16-19). Data are shown asmean±SEM. P value is indicated when two conditions were compared with anunpaired two-tailed Student's t-test (*: P<0.05).

FIG. 11 is a histogram showing that the administration of either live orpasteurized A. muciniphila significantly decreases serum HDL-cholesteroland lead to a similar trend for LDL-cholesterol. Plasma VLDL, LDL andHDL cholesterol levels determined by fast protein liquid chromatography(FPLC). Mice were fed a control diet (CT ND), a high-fat diet (CT HFD)and treated daily by oral gavage with sterile anaerobic PBS containingglycerol or A. muciniphila grown on a non-mucus-based medium, eitherlive (HFD Akk G), or pasteurized (HFD Akk P) for 5 weeks (n=8-10). Datawith different superscript letters are significantly different (P<0.05),according to two-way ANOVA analysis followed by Bonferonni post-test.

FIG. 12 is a histogram showing that the administration of pasteurized A.muciniphila increases energy excreted in the feces. Fecal energymeasured by indirect bomb calorimetry (kcal/g feces) (n=5). Mice werefed a control diet (CT ND), a high-fat diet (CT HFD) and treated dailyby oral gavage with sterile anaerobic PBS containing glycerol or A.muciniphila grown on a non-mucus-based medium, either live (HFD Akk G),or pasteurized (HFD Akk P) for 5 weeks. Data are shown as mean±SEM. Datawith different superscript letters are significantly different (P<0.05),according to one-way ANOVA analysis followed by Tukey post-hoc test.

FIG. 13 is a graph showing that the administration of pasteurized A.muciniphila induces a larger correction of the HFD-induced shift in hosturinary metabolomics profile than live A. muciniphila. (a) OrthogonalPartial Least Squares discriminant analysis (OPLS-DA) score plots forurine metabolic profiles (n=5-7). (b) Impact of all treatments on thepredictive component 1 of the OPLS-DA analysis. Mice were fed a controldiet (CT ND), a high-fat diet (CT HFD) and treated daily by oral gavagewith sterile anaerobic PBS containing glycerol or A. muciniphila grownon a non-mucus-based medium, either live (HFD Akk G), or pasteurized(HFD Akk P) for 5 weeks.

FIG. 14 is a set of graphs showing the safety assessment of A.muciniphila after oral administration in overweight/obese patients(n=5). (A-C) Markers related to inflammation and hematology:(A)C-reactive protein (mg/dl), (B) Total white blood cell count (10³cells/μL), (C) Prothrombin time (sec). (D-F) Markers related to kidneyfunction: (D) Urea (mg/dl), (E) Creatinine (mg/dl), (F) Glomerularfiltration rate (mL/min*1.73 m²). (G-I) Markers related to liverfunction: (G) Alanine transaminase activity (IU/l), (H) Aspartatetransaminase activity (IU/l), (I) γ-glutamyltranspeptidase activity(IU/l). (J-K) Markers related to muscle function: (J) Creatinine kinaseactivity (IU/l), (K) Lactate dehydrogenase activity (IU/l).

EXAMPLES

The present invention is further illustrated by the following examples.

We previously showed that daily administration of Akkermansiamuciniphila to mice fed a high-fat diet can impede the development ofobesity (WO 2014/076246).

With the perspective of transferring these results to clinical settings,we decided to assess whether A. muciniphila would retain its effectswhen cultured on a non-mucus-based medium suited for human trials.Moreover, our previous results indicated that autoclaving A. muciniphilaabolished its effect on diet-induced obesity. We therefore sought toinvestigate the consequences of another inactivation method (i.e.pasteurization) on A. muciniphila-mediated effects.

Materials and Methods Mice

First experiment: a set of 10-week-old C57BL/6J mice (50 mice,n=10/group) (Charles River, L'Arbresle, France) were housed in acontrolled environment (12 h daylight cycle, lights off at 6 μm) ingroups of two mice per cage, with free access to food and water. Micewere fed a control diet (ND) (AIN93Mi, Research diet, New Brunswick,N.J., USA) or a high-fat diet (HFD) (60% fat and 20% carbohydrates(kcal/100 g) D12492i, Research diet, New Brunswick, N.J., USA).

Mice were treated daily with an oral administration of Akkermansiamuciniphila grown on a mucin-based medium (HFD Akk M) or anon-mucus-based medium (HFD Akk G) by oral gavage at the dose of 2.10⁸cfu/0.15 mL suspended in sterile anaerobic phosphate buffer saline(PBS). Additionally, one group of mice was treated daily with an oraladministration of Akkermansia muciniphila grown on a non-mucus-basedmedium and inactivated by pasteurization (HFD Akk P). Control groupswere treated with an oral gavage of an equivalent volume of sterileanaerobic PBS (CT ND and CT HFD) containing a similar end concentrationof glycerol (2.5% vol/vol). Treatment was continued for 4 weeks.

For the HFD Akk M group, A. muciniphila MucT (ATTC BAA-835) was grownanaerobically in a mucin-based basal medium as previously described(Derrien et al., 2004. Int. J. Syst. Evol. Microbiol. 54:1469-1476).Cultures were then washed and suspended in anaerobic PBS, including 25%(v/v) glycerol, to an end concentration of 1.10¹⁰ cfu/mL.

For the HFD Akk G group, A. muciniphila MucT (ATTC BAA-835) was grownanaerobically in non-mucus-based medium. Cultures were then washed andsuspended in anaerobic PBS, including 25% (v/v) glycerol, to an endconcentration of 1.10¹⁰ cfu/mL.

For the HFD Akk P group, A. muciniphila MucT (ATTC BAA-835) was grownanaerobically in non-mucus-based medium Cultures were then washed andsuspended in anaerobic PBS, including 25% (v/v) glycerol, to an endconcentration of 1.10¹⁰ cfu/mL. Vials were then pasteurized by exposureto a temperature of 70° C. for 30 minutes in a water bath.

Body weight, food and water intake were recorded once a week. Bodycomposition was assessed by using 7.5 MHz time domain-nuclear magneticresonance (TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).

Second experiment: a set of 10-week-old C57BL/6J mice (40 mice,n=10/group) (Charles River, L'Arbresle, France) were housed in acontrolled environment (12 h daylight cycle, lights off at 6 μm) ingroups of two mice per cage, with free access to food and water. Micewere fed a control diet (ND) (AIN93Mi; Research diet, New Brunswick,N.J., USA) or a high-fat diet (HFD) (60% fat and 20% carbohydrates(kcal/100 g), Research diet D12492i, New Brunswick, N.J., USA). Micewere treated daily with an oral administration of Akkermansiamuciniphila grown on a non-mucus-based medium and either live orpasteurized (HFD Akk G and HFD Akk P) by oral gavage at the dose of2.10⁸ cfu/0.15 mL suspended in sterile anaerobic phosphate buffersaline. Control groups were treated with an oral gavage of an equivalentvolume of sterile anaerobic phosphate buffer saline (CT ND and CT HFD).Treatment was continued for 5 weeks.

For the HFD Akk G group, A. muciniphila MucT (ATTC BAA-835) was grownanaerobically in non-mucus-based medium. Cultures were then washed andsuspended in anaerobic PBS, including 25% (v/v) glycerol, to an endconcentration of 1.10¹⁰ cfu/mL.

For the HFD Akk P group, A. muciniphila MucT (ATTC BAA-835) was grownanaerobically in non-mucus-based medium. Cultures were then washed andsuspended in anaerobic PBS, including 25% (v/v) glycerol, to an endconcentration of 1.10¹⁰ cfu/mL. Vials were then pasteurized by exposureto a temperature of 70° C. for 30 minutes in a water bath.

Body weight, food and water intake were recorded once a week. Bodycomposition was assessed by using 7.5 MHz time domain-nuclear magneticresonance (TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).

Fresh urinary samples were collected during the final week of treatmentand directly stored at −80° C. before analysis. Fecal energy content wasmeasured on fecal samples harvested after a 24 h period during the finalweek of treatment by the use of a bomb calorimeter (Mouse ClinicalInstitute, 67404 Illkirch, France).

Third experiment: a set of 10-week-old C57BL/6J mice (40 mice,n=10/group) (Charles River, L'Arbresle, France) were housed in acontrolled environment (12 h daylight cycle, lights off at 6 μm) ingroups of two mice per cage, with free access to food and water. Micewere fed a control diet (CT ND) (AIN93Mi; Research diet, New Brunswick,N.J., USA) or a high-fat diet (CT HFD) (60% fat and 20% carbohydrates(kcal/100 g), Research diet D12492i, New Brunswick, N.J., USA). Micewere treated daily with an oral administration of Akkermansiamuciniphila grown on a non-mucus-based medium and either live orpasteurized (HFD Akk G and HFD Akk P) by oral gavage at the dose of2.10⁸ CFU/0.15 mL suspended in sterile anaerobic phosphate buffersaline. Control groups were treated with an oral gavage of an equivalentvolume of sterile anaerobic phosphate buffer saline (CT ND and CT HFD).Treatment was continued for 5 weeks.

All mouse experiments were approved by and performed in accordance withthe guidelines of the local ethics committee. Housing conditions werespecified by the Belgian Law of May 29, 2013, regarding the protectionof laboratory animals (agreement number LA1230314).

Oral Glucose Tolerance Test

6 h-fasted mice were treated with an oral gavage glucose load (2 gglucose per kg body weight). Blood glucose levels were measured beforeoral glucose load and 15, 30, 60, 90 and 120 minutes after oral glucoseload. Blood glucose was determined with a glucose meter (Accu Check,Aviva, Roche) on blood samples collected from the tip of the tail vein.

Insulin Resistance Index

Plasma insulin concentration was determined in 5 μL of plasma using anELISA kit (Mercodia) according to the manufacturer's instructions.Insulin resistance index was determined by multiplying the area underthe curve of both blood glucose (−30 to 120 minutes) and plasma insulin(−30 and 15 minutes) obtained following the oral glucose tolerance test.

Western-Blot

To analyze the insulin signaling pathway in the third experiment, micewere allocated in either a saline-injected subgroup or aninsulin-injected subgroup so that both subgroups were matched in termsof body weight and fat mass. They then received 1 mU/g insulin(Actrapid; Novo Nordisk A/S, Denmark) under anaesthesia (isoflurane,Forene, Abbott, Queenborough, Kent, England), or an equal volume ofsaline solution into the portal vein. Three minutes after injection,mice were killed and liver was rapidly harvested.

For detection of proteins of the insulin pathway, tissues werehomogenized in ERK buffer (Triton X-100 0.1%, HEPES 50 mM, NaCl 5 M,Glycerol 10%, MgCl₂ 1.5 mM and DTT 1 mM) supplemented with a cocktail ofprotease inhibitors and phosphatase inhibitors. Equal amounts ofproteins were separated by SDS-PAGE and transferred to nitrocellulosemembranes. Membranes were incubated overnight at 4° C. with antibodiesdiluted in Tris-buffered saline Tween-20 containing 1% non-fat drymilk:p-IRb (1:1,000; sc-25103, Santa Cruz, Calif., USA), p-AktThr308(1:1.000; #2965L, Cell Signaling, Danvers, Mass., USA) and p-AktSer473(1:1.000; #4060L, Cell Signaling). Quantification of phosphoproteins wasperformed on 5 animals with insulin injection and 5 animals with salineinjection per group. The loading control was β-actin (1:10000; ab6276).

Tissue Sampling

The animals have been anesthetized with isoflurane (Forene®, Abbott,Queenborough, Kent, England) and blood was sampled from the portal andcava veins. Mice were then killed by cervical dislocation beforeproceeding to tissue sampling. Adipose depots (epididymal, subcutaneousand mesenteric) were precisely dissected and weighed; the addition ofthe weights of all three adipose tissue depots corresponds to theadiposity index. The intestinal segments (ileum, cecum and colon), cecalcontent and adipose tissue depots were immersed in liquid nitrogen, andstored at −80° C., for further analysis.

Histological Analyses

Adipose tissues were fixed in 4% paraformaldehyde for 24 hours at roomtemperature. Then the samples were immersed in ethanol 100% for 24 hoursprior to processing for paraffin embedding. Tissue samples, paraffinsections of 5 μm, were stained with hacmatoxylin and eosin. Images wereobtained using the SCN400 slide scanner (Lcica Biosystcms, Wetzlar,Germany). 5 high-magnification fields were selected at random for eachmouse and adipocyte diameter was determined using ImageJ (Version 1.50a,National Institutes of Health, Bethesda, Md., USA).

RNA Preparation and Real-Time qPCR Analysis

Total RNA was prepared from tissues using TriPure reagent (Roche).Quantification and integrity analysis of total RNA was performed byrunning 1 μL of each sample on an Agilent 2100 Bioanalyzer (Agilent RNA6000 Nano Kit, Agilent). cDNA was prepared by reverse transcription of 1μg total RNA using a Reverse Transcription System kit (Promega, Leiden,The Netherlands). Real-time PCRs were performed with the Biorad CFXreal-time PCR system and software (Biorad, Hercules, United States)using Mesa Fast qPCR (Eurogentec, Seraing, Belgium) for detectionaccording to the manufacturer's instructions. RPL19 was chosen as thehousekeeping gene. All samples were run in duplicate in a single 96-wellreaction plate, and data were analyzed according to the 2^(ΔΔCT) method.The identity and purity of the amplified product was checked throughanalysis of the melting curve carried out at the end of amplification.Primer sequences for the targeted mouse genes are presented in Table 1below.

TABLE 1 Primer sequences for the targeted mouse genes. Primers SequenceRPL-19 Forward GAAGGTCAAAGGGAATGTGTTCA (SEQ ID NO: 1) ReverseCCTTGTCTGCCTTCAGCTTGT (SEQ ID NO: 2) Ocln Forward ATGTCCGGCCGATGCTCTC(SEQ ID NO: 3) Reverse TTTGGCTGCTCTTGGGTCTGTAT (SEQ ID NO: 4) Cldn3Forward TCATCGGCAGCAGCATCATCAC (SEQ ID NO: 5) ReverseACGATGGTGATCTTGGCCTTGG (SEQ ID NO: 6) Lyz1 ForwardGCCAAGGTCTACAATCGTTGTGAGTTG (SEQ ID NO: 7) ReverseCAGTCAGCCAGCTTGACACCACG (SEQ ID NO: 8)

Measurement of Plasma Triglycerides

Plasma samples were assayed for triglycerides by measuring the glycerolresulting from hydrolysis of triglycerides, using a commercial kit(DiaSys, Condom, France).

Measurement of Plasma Leptin

Plasma samples were assayed for leptin through the use of a multipleximmunoassay kit (Merck Millipore, Brussels, Belgium) and measured usingLuminex technology (Bioplex, Bio-Rad, Belgium) following themanufacturer's instructions.

Measurement of Plasma Cholesterol (Fast Protein Liquid Chromatography,FLPC)

Quantification of plasma lipoproteins was performed using fast proteinliquid chromatography (FPLC, AKTA purifier 10, GE Healthcare, Chicago,Ill., USA). 50 μL of individual plasma was injected and lipoproteinswere separated on Superose™ 6 10/300 GL column (GE Healthcare, Chicago,Ill., USA) with NaCl 0.15 M at pH 7.4 as mobile phase at a 1 mL/min flowrate. The effluent was collected into fractions of 0.3 mL thencholesterol and TG content in each fraction were determined as describedabove. Quantification of cholesterol in lipoprotein classes (VLDL, LDL,and HDL) was performed by measuring the percentage peak area and bymultiplying each percentage to the total amount of cholesterol. Plasmatotal cholesterol was measured with commercial kits (CHOD-PAP; BIOLABOSA, Maizy, France).

Measurement of Fecal Energy

Fecal energy content was measured on fecal samples harvested after a 24h-period during the final week of treatment by the use of a bombcalorimeter (Mouse Clinical Institute, Illkirch, France).

Urinary Metabolomics Analyses

Mouse urine samples were prepared and measured on a spectrometer(Bruker) operating at 600.22 MHz 1H frequency according to previouslypublished protocol (Dona A C, 2014); the ¹H NMR spectra were thenprocessed and analyzed as described previously (Dumas et al., 2006.Proc. Natl. Acad. Sci. USA. 103(33):12511-6).

Quantification of Plasma Lipopolysaccharide

Portal vein blood LPS concentration was measured using anEndosafe-Multi-Cartridge System (Charles River Laboratories) based onthe Limulus amacbocyte lysate (LAL) kinetic chromogenic methodology thatmeasures color intensity directly related to the endotoxin concentrationin a sample. Plasmas were diluted 1/10 with endotoxin-free buffer tominimize interferences in the reaction (inhibition or enhancement) andheated 15 minutes at 70° C. Each sample was diluted 1/100, 1/150, 1/200or 1/400 with endotoxin-free LAL reagent water (Charles RiverLaboratories) and treated in duplicate, and two spikes for each samplewere included in the determination. All samples have been validated forthe recovery and the coefficient variation. The lower limit of detectionwas 0.005 EU/mL.

Determination of the Pasteurization Temperature and Time Range

Vials containing live bacteria were immersed in a water bath set to 50,60, 70, 80 or 90° C. for 15 seconds (0.25 minutes), 2 minutes, 5minutes, 15 minutes and 30 minutes. Inactivation of A. muciniphila wasassessed by plating 50 μL of undiluted vial content on Brain-HeartInfusion (BHI)-Agar medium supplemented with 5% mucus and looking forthe presence of colony-forming units (cfu) after 7 days of incubation at37° C. in an anaerobic container. Content of an autoclaved vial was usedas a negative control, and content from a vial non immersed in a waterbath was used as a positive control. This experiment was performed attwo different times.

Mucus-Based Medium

A. muciniphila was grown in mucus-based medium, washed and concentratedas described previously (Everard et al., 2013. Proc. Natl. Acad. Sci.USA. 110:9066-9071). In addition to an untreated batch of cells, onepart was subject to a mild heat treatment by a 30-minute incubation at70° C.

Non-Mucus-Based Medium

A. muciniphila was grown in a non-mucus-based medium consisting of basalanaerobic medium as described previously (Derrien et al., 2004. Int. J.Syst. Evol. Microbiol. 54:1469-1476) containing 16 g/L soy-based pepton,25 mM glucose and 25 mM N-acetyl-glucosamine and 4 g/L L-threonine. Thecells were washed and concentrated as described previously (Everard etal., 2013. Proc. Natl. Acad. Sci. USA. 110:9066-9071). In addition to anuntreated batch of cells, one part was subject to a mild heat treatmentby a 30-minute incubation at 70° C.

Safety Assessment of Oral Administration of Live and Pasteurized A.muciniphila in Overweight or Obese Volunteers

Results presented are interim safety reports from twenty overweight andobese patients (Body mass index >25 kg/m²) presenting a metabolicsyndrome following the NCEP ATP III definition (any three of the fivefollowing criteria: fasting glycaemia >110 mg/dL, blood pressure ≥130/85mm Hg or antihypertensive treatment, fasting triglyceridemia ≥150 mg/dL,HDL cholesterol <40 mg/dL for males, 50 mg/dL for females, and/or waistcircumference >102 cm for males, 88 cm for females). Patients werevoluntarily recruited from the Cliniques Universitaires Saint Luc,Brussels, Belgium between December 2015 and May 2016. Subjects wereassigned to any of the treatment arms following a randomized blockdesign. The exclusion criteria were: presence of acute or chronicprogressive or chronic unstabilized diseases, alcohol consumption (>2glasses/day), previous bariatric surgery, any surgery in the 3 monthsprior to the study or planned in the next 6 months, pregnancy orpregnancy planned in the next 6 months, regular physical activity (>30minutes of sports 3 times a week), consumption of dietary supplements(omega-3 fatty acids, probiotics, prebiotics, plant stanols/sterols) inthe month prior the study, inflammatory bowel disease or irritable bowelsyndrome, diabetic gastrointestinal autonomic neuropathy (such asgastroparesis or reduced gastrointestinal motility), consumption of morethan 30 g of dietary fibers per day, consumption of vegetarian orunusual diet, lactose intolerance or milk protein allergy, glutenintolerance, current treatment with medications influencing parametersof interest (glucose-lowering drugs such as metformin, DPP-4 inhibitors,GLP-1 receptor agonists, acarbose, sulfonylueras, glinides,thiazolidinediones, SGLT2 inhibitors, insulin, lactulose, consumption ofantibiotics in the 2 months prior the study, glucocorticoids,immunosuppressive agents, statins, fibrates, orlistat, cholestyramine,or ezetimibe), and baseline glycated hemoglobin (HbA1c) >7.5%. TheCommission d'Ethique Biomédicale Hospitalo-facultaire from theUniversité catholique de Louvain (Brussels, Belgium) provided ethicalapproval for this study and written informed consent was obtained fromeach participant. The trial was registered at clinicaltrials.gov asNCT02637115.

Subjects were assigned to receive either a daily dose of placebo (anequivalent volume of sterile PBS containing glycerol), 10¹⁰ CFU live A.muciniphila (Akk S-10¹⁰), 10⁹ CFU live A. muciniphila (Akk S-10⁹), or10¹⁰ CFU pasteurized A. muciniphila (Akk P-10¹⁰) (placebo and bacteriawere produced at a food-grade level according to good manufacturingpractices) for 3 months. Blood samples were collected at the beginningof the treatment and a portion was directly sent to the hospitallaboratory to measure relevant clinical parameters. Different tubes wereused based on the clinical parameter: EDTA-coated tubes for white bloodcell count, Sodium fluoride-coated tubes for fasting glycemia,citrate-coated tubes for clotting assays, and lithium-heparin-coatedtubes for urea and enzymatic activities. After 2 weeks of treatment,patients came back to the hospital for a safety visit, where bloodsamples were collected to allow comparison of clinical parameters tobaseline values.

The patients and the physicians were blinded to the treatment. For FIG.14 and Tables 3-5, the number of subjects per group is: Placebo: 5, AkkS-10¹⁰: 5, Akk S-10⁹: 5, Akk P-10¹⁰: 5.

Statistical Analysis

Data are expressed as means±SEM. Differences between two groups wereassessed using the unpaired two-tailed Student's t-test. Data setsinvolving more than two groups were assessed by ANOVA followed by Tukeypost-hoc tests. Data with different superscript letters aresignificantly different with P<0.05, according to the post-hoc ANOVAstatistical analysis. Data were analyzed using GraphPad Prism version5.00 for windows (GraphPad Software, San Diego, Calif., USA). Resultswere considered statistically significant when P<0.05.

A two-way ANOVA analysis with a Bonferonni post-test on repeatedmeasurements was performed for the evolution of glycemia during theOGTT, for the reparation cholesterol in specific lipoproteins and forwestern-blot analyses.

Human data are expressed as the mean±SD. Differences between groups wereassessed using Kruskal-Wallis test. Differences between values observedat baseline and at the time of the safety visit were assessed using aWilcoxon matched-pairs signed rank test. Data were analyzed usingGraphPad Prism version 7.00 for Windows (GraphPad Software, San Diego,Calif., USA). The results were considered statistically significant whenp<0.05.

Results In Vitro Experiments

In order to optimize the pasteurization protocol, we first incubatedvials containing A. muciniphila in water baths set to a range oftemperature for different times. Pasteurization was considered effectivewhen no bacteria could be observed after plating the treated vialcontents on a rich medium (Table 2).

TABLE 2 Combinations of temperatures and exposure times tested forpasteurization. “Live” corresponds to plates where cfu were obtained inhigh numbers. “Borderline”corresponds to plates where between 1 and 3cfu were observed. “Inactivated” corresponds to plates where no cfucould be observed. Temperature (° C.) 50 60 70 80 90 Exposure 0.25 LiveLive Live Live Live (minutes) 2 Live Live Live Inactivated Inactivated 5Live Live Borderline Inactivated Inactivated 15 Live BorderlineBorderline Inactivated Inactivated 30 Borderline Inactivated InactivatedInactivated InactivatedFor the further experiments, we have selected a pasteurization of 30minutes at 70° C. In addition to the viability, the effect ofpasteurization has been tested on the activity of two A. muciniphilafucosidases and 2 sulfatases (encoded by the genes Amuc_0010, Amuch_0146and Amuc_0121 and Amuc_1074; van Passel et al., 2011. PLoS One.6(3):e16876). These enzymes are relevant for the degradation of mucin.For this purpose, their genes were overexpressed in Escherichia coli asdescribed with a C-terminal His-tag (Tailford et al., 2015. Nat. Commun.6:7624) and the purified proteins were used for the analysis. The enzymeactivities were determined before and after 30 minutes at 70° C. andthis treatment completely resulted in an over 20-fold inactivation ofthe enzymatic activities.

In Vivo Experiments

In a first set of experiments, mice fed a high-fat diet were treateddaily with an oral gavage of live A. muciniphila grown either on amucus-based or a non-mucus-based medium. Another group of mice wastreated with an oral gavage of A. muciniphila grown on a non-mucus-basedmedium and inactivated by pasteurization (30 minutes at 70° C.). Micefed standard chow were used as a control group. Treatment was carried onfor 4 weeks.

We observed that live A. muciniphila treatment reduced high-fat dietinduced body weight and fat mass gain, regardless of the growth mediumused (FIG. 1a-b ). Surprisingly, pasteurized A. muciniphila exerted astronger effect than the live bacterium, as mice treated withpasteurized cells showed a similar body weight gain and fat mass gain tomice fed a control diet (FIG. 1a-b ). Adiposity index, shown as the sumof subcutaneous, visceral and epididymal adipose tissue depots, wassignificantly increased in mice fed a high-fat diet (FIG. 2).Administration of A. muciniphila counteracted this increase, to asimilar extent regardless of the growth medium or pasteurization.

We next confirmed our previous results in terms of glucose tolerance.Indeed, a high-fat diet leads to increased glycaemia following an oralglucose tolerance test (OGTT), resulting in a significantly higher areaunder the curve (AUC) measured between 30 minutes before and 120 minutesafter glucose administration (FIG. 3a-b ). Administration of A.muciniphila mitigated this increase, leading to intermediary AUC values,once again independently of the growth medium and pasteurization.

When taking into account insulinemia of the mice, the insulin resistanceindex of mice fed a high-fat diet was significantly higher than forcontrol mice (FIG. 3c ). Treatment with A. muciniphila grown on amucus-based medium resulted in intermediary insulin resistance (IR)index values between control and untreated high-fat diet-fed mice.However, although the IR index of mice treated with A. muciniphila grownon a non-mucus-based medium was 15% lower than for untreated mice fed ahigh-fat diet, it was still significantly higher than for control mice,while pasteurized A. muciniphila completely normalized the IR index ofmice fed a high-fat diet (FIG. 3c ), thereby showing that pasteurizationincreases the effects of Akkermansia muciniphila on glucose toleranceand insulin resistance.

We previously found that treatment with A. muciniphila could impact gutbarrier function through modulation of antimicrobial peptides productionand regulation of mucus layer thickness. To further increase ourunderstanding of the cross-talk between A. muciniphila and theintestinal barrier, we measured the expression of two markers ofintestinal tight junction proteins, namely Ocln and Cldn3, encoding theproteins Occludin and Claudin 3; as well as Lyz1 encoding theantimicrobial peptide Lysozyme 1. In the jejunum, treatment of HFD-fedmice with live or pasteurized A. muciniphila increased the expression ofOcln, while pasteurized A. muciniphila specifically increased Lyz1expression (FIG. 4a ). In the ileum, treatment with live and pasteurizedA. muciniphila increased the expression of Cldn3 and Lyz1 (FIG. 4b ).These effects on markers of intestinal integrity resulted in a completenormalization of plasma LPS in treated mice (FIG. 4c ), showing thatboth the live and pasteurized form of A. muciniphila can strengthen theintestinal barrier and decrease metabolic endotoxemia.

In a second and third set of experiments, we treated high-fat diet micewith A. muciniphila grown on the non-mucus-based medium, either live orpasteurized, to confirm the effects obtained above. Mice fed standardchow were used as a control group, and treatment was carried on for fiveweeks. Treatment with A. muciniphila grown on non-mucus-based mediumlead to a 10 to 15% decrease of body weight gain, fat mass gain andadiposity index in mice fed a high-fat diet, although without reachingstatistical significance (FIGS. 5 and 6). Administration of pasteurizedA. muciniphila completely normalized these parameters, once againshowing a stronger effect following pasteurization.

We also obtained similar results in terms of glucose tolerance andinsulin sensitivity. Indeed, while untreated mice fed a high-fat dietexhibited a higher AUC during the course of the OGTT (FIG. 7a-b ),treatment with live or pasteurized A. muciniphila normalized thisparameter. The IR index of mice treated with A. muciniphila grown on anon-mucus-based medium was 20% lower than for untreated high-fatdiet-fed mice, but still significantly higher than for control mice.However, treatment with pasteurized A. muciniphila completely normalizedthe IR index with a two-fold decrease when compared to the untreatedhigh-fat diet-fed group (FIG. 7c ).

In the third set of experiments, while untreated mice fed a high-fatdiet exhibited a higher AUC during the course of the OGTT, treatmentwith live or pasteurized A. muciniphila significantly decreased the AUC,showing an improvement of glucose tolerance (FIG. 8 a-b). In order tofurther investigate the effects of A. muciniphila on insulinsensitivity, in addition to the OGTT, we analyzed insulin-inducedphosphorylation of the insulin receptor (IR) and its downstream mediatorAkt in the liver at the threonine (Akt^(thr)) and serine (Akt^(ser))sites after insulin or saline solution injection in the portal vein(FIG. 8 c-e). As previously described, untreated high-fat diet-fed micedisplayed decreased phosphorylation of all assessed proteins whencompared to CT ND mice, reaching significance for Akt^(thr) (FIG. 8d ).Treatment with A. muciniphila tended to counteract these effects, withsignificantly higher levels of p-Akt^(ser) in mice treated with the livebacterium (FIG. 8e ) when compared to untreated HFD-fed mice.

We then measured the mean adipocyte diameter in the subcutaneous adiposedepot, as it is known to be increased in obesity and to contribute tothe development of inflammation and insulin resistance (Rosen andSpiegelman, 2014. Cell. 156:20-44). In accordance with the literature,we observed that a high-fat diet leads to an increased diameter.Treatment with live A. muciniphila grown on a non-mucus-based did notaffect the high-fat diet-induced-increased diameter. However,administration of pasteurized A. muciniphila restored the diameter tosimilar levels as in control mice (FIG. 9a-b ). Treatment withpasteurized A. muciniphila also normalized leptin concentration tosimilar levels as observed in control mice (FIG. 9c ).

The next parameter analyzed concerned the dyslipidemia induced byhigh-fat diet feeding. We assessed the effects of A. muciniphila onhypertriglyceridemia and hypercholesterolemia, which is associated withatherosclerosis and cardiovascular disease. Although no difference couldbe observed between control and untreated high-fat diet-fed mice, weobserved that treatment with pasteurized A. muciniphila leads to asignificant reduction (between 15 and 20%) of plasma triglyceride levels(FIG. 10). Regarding plasma cholesterol, treatment with either live orpasteurized A. muciniphila corrected the HFD-inducedhypercholesterolemia, with significant decreases in plasmaHDL-cholesterol and a similar trend for LDL-cholesterol (FIG. 11).

To further explain how live and pasteurized A. muciniphila reduce bodyweight and fat mass gain without affecting food intake on a high-fatdiet, we measured fecal caloric content and found that it wassignificantly increased in mice treated with pasteurized A. muciniphilabut not with live A. muciniphila (FIG. 12). These results suggested adecrease in energy absorption and therefore energy excretion in thefeces following pasteurized A. muciniphila administration, which could,at least in part, explain the greater effects observed with pasteurizedA. muciniphila.

We next assessed whether treatment with A. muciniphila could reduce theHFD-induced shift in the host urinary metabolome (FIG. 13). High-fatdiet was the main factor influencing 1H NMR-based untargeted metabolicprofiles on the first O-PLS-DA score (Tpred1) while treatment withpasteurized A. muciniphila clustered separately from all other groupsregarding the second score (Tpred2, FIG. 13). This resulted in anormalization of the HFD-induced shift of 37% with the pasteurizedbacterium, and 17% with the live bacterium (FIG. 13).

Altogether, these data suggest that the effects of A. muciniphila onhost metabolism arc mostly similar regardless of the growth medium used.More surprisingly, they also show that pasteurization potentiates theeffects of A. muciniphila. This is of utmost interest as pasteurizationcould decrease biosafety issues associated with the use of a livebacterium while increasing the efficacy of A. muciniphila in thetreatment of obesity and associated disorders.

Safely Assessment of Oral Administration of Live and Pasteurized A.muciniphila in Overweight or Obese Volunteers

We evaluated the safety and tolerability of A. muciniphila oraladministration in overweight and obese volunteers treated with differentdoses of live A. muciniphila (Akk S-10¹⁰ and Akk S-10⁹) or pasteurizedA. muciniphila (Akk P-10¹⁰) as part of an ongoing clinical study testingthe efficacy of this bacterium against the metabolic syndrome.Anthropomorphic characteristics of the patients at the beginning of theintervention are reported in Table 3.

TABLE 3 Descriptive characteristics at the beginning of treatment forall subjects included in the clinical study (n = 5) Placebo Akk S - 10¹⁰Akk S - 10⁹ Akk P - 10¹⁰ Sex (M/W) 1/4 3/2 2/3 2/3 Age (Years)  53.00 ±10.98 50.40 ± 4.72 50.60 ± 6.69 52.40 ± 7.99 Body weight (Kg) 102.60 ±13.53 111.10 ± 19.52 103.80 ± 17.03 122.50 ± 12.67 Body mass index 35.84± 5.98 38.48 ± 5.37 36.30 ± 3.12 40.71 ± 5.71 (Kg/m²) Waist 116.60 ±13.03 119.50 ± 12.35 115.60 ± 7.20  124.90 ± 8.10  circumference (cm)Fasting glycaemia 100.50 ± 10.52 96.13 ±2.24  108.30 ± 12.91 106.30 ±11.80 (mg/dl)

We analyzed several clinical parameters investigated in probioticssafety assessments (Jones et al., 2012. Food. Chem. Toxicol.50:2216-2223; Burton et al., 2011. Food Chem. Toxicol. 49(9):2356-64;Wind et al., 2010. Br. J. Nutr. 104(12):1806-16) before and two weeksafter starting the treatment. No significant changes on markers relatedto inflammation and hematology, kidney, liver and muscle function wereobserved with any formulation of A. muciniphila (FIGS. 14A-K and Table4).

TABLE 4 Descriptive characteristics at the beginning of treatment forall subjects included in the clinical study (n = 5) Placebo Akk S-10¹⁰Akk S-10⁹ Akk P-10⁹ Baseline Safety Baseline Safety Baseline SafetyBaseline Safety Inflammation & Hematology C-reactive  3.60 ±  4.40 ± 6.60 ±  6.40 ±  6.60 ±  6.40 ± 11.40 ± 15.20 ± protein 1.67 2.07 5.186.07 5.18 6.07 14.33 17.38 (mg dl⁻¹) White blood  6.43 ±  7.07 ±  7.91 ± 8.36 ±  7.91 ±  8.36 ±  6.89 ±  8.20 ± cells (10³ μL⁻¹) 1.49 1.68 4.084.17 4.08 4.17 2.44 1.61 Prothrombin 11.38 ± 11.14 ± 10.92 ± 11.12 ±10.92 ± 11.12 ± 11.28 ± 11.20 ± time (sec) 0.55 0.44 0.73 0.80 0.73 0.800.56 0.56 Liver enzymes Alanine 24.00 ± 23.20 ± 27.40 ± 24.40 ± 27.40 ±24.40 ± 29.20 ± 27.80 ± Aminostrans- 14.82 15.71 27.32 13.85 27.32 13.8513.72 12.05 ferase activity (IU l⁻¹) Aspartate 17.00 ± 16.60 ± 19.33 ±17.67 ± 19.33 ± 17.67 ± 23.00 ± 19.80 ± Aminotrans- 6.33 6.35 9.48 5.059.48 5.05 9.14 7.98 ferase activity (IU l⁻¹) γ-Glutamyl- 22.40 ± 23.60 ±40.40 ± 33.40 ± 40.40 ± 33.40 ± 45.20 ± 42.80 ± transferase 15.76 18.0538.44 24.42 38.44 24.42 28.90 24.94 activity (IU l⁻¹) Kidney functionUrea (mg dl⁻¹) 35.20 ± 30.00 ± 28.60 ± 30.40 ± 28.60 ± 30.40 ± 31.40 ±43.40 ± 10.26 7.25 9.42 4.98 9.42 4.98 2.88 18.96 Creatinine  0.73 ± 0.71 ±  0.78 ±  0.80 ±  0.78 ±  0.80 ±  0.83 ±  0.89 ± (mg dl⁻¹) 0.110.10 0.09 0.15 0.09 0.15 0.18 0.21 Glomerular 92.20 ± 95.20 ± 88.60 ±88.60 ± 88.60 ± 88.60 ± 83.80 ± 78.00 ± filtration rate 22.52 17.1110.06 20.19 10.06 20.19 14.17 15.41 (mL min⁻¹ 1.73 m⁻²) Muscle enzymesCreatinine 78.80 ± 79.40 ± 92.40 ± 94.80 ± 92.40 ± 94.80 ± 162.40 ± 135.50 ±  Kinase activity 25.37 28.06 40.32 38.11 40.32 38.11 122.3087.53 (IU l⁻¹) Lactate 176.60 ±  167.20 ±  172.60 ±  176.20 ±  172.60 ± 176.20 ±  180.60 ±  171.40 ±  Dehydrogenase 19.86 22.86 20.74 33.2220.74 33.22 17.70 34.44 activity (IU l⁻¹)

Moreover, the frequency of recorded adverse effects was similar in allgroups (Table 5).

TABLE 5 Proportion of patients experiencing self-reported adverseeffects (n = 5) Placebo Akk S - 10¹⁰ Akk S - 10⁹ Akk P - 10⁹ Nausea 1/5 0 2/5 1/5 Flatulence  0 1/5 3/5 1/5 Bloating 1/5 1/5  0  0 Cramps 1/51/5  0 1/5 Borborygmi  0 3/5 3/5  0 Gastric reflux 1/5  0 1/5  0

Borborygmi were reported by some patients treated with live A.muciniphila, but the difference with other groups was not significant.

While the number of subjects is limited, these first human data suggestthat both live and pasteurized A. muciniphila are well tolerated inobese/overweight volunteers and appear safe for oral administration.

Furthermore, promising trends were observed in terms of fat mass,glycemia and inflammation markers at the end of the treatment period forpatients treated with the high dose of live and/or pasteurized A.muciniphila.

1. A method for treating a metabolic disorder in a subject in need thereof, comprising administering to the subject Akkermansia muciniphila or fragments thereof, wherein the Akkermansia muciniphila is pasteurized.
 2. The method of claim 1, wherein said metabolic disorder is obesity.
 3. The method of claim 1, wherein said metabolic disorder is selected from the group consisting of: metabolic syndrome; insulin-deficiency or insulin-resistance related disorders; diabetes mellitus including type 2 diabetes; glucose intolerance; abnormal lipid metabolism; atherosclerosis; hypertension; pre-eclampsia; cardiac pathology; stroke; non-alcoholic fatty liver disease; hyperglycemia; hepatic steatosis; liver diseases including fibrosis associated with obesity and abnormal liver functions, more particularly changes in bile production and immunity; dyslipidemia; dysfunction of the immune system associated with overweight and obesity; inflammatory, immune and barrier function diseases including inflammatory bowel disease, more particularly Crohn's disease and ulcerative colitis, and irritable bowel syndrome; cardiovascular diseases; high cholesterol; elevated triglycerides; asthma; sleep apnea; osteoarthritis; neuro-degeneration; gallbladder disease; syndrome X; atherogenic dyslipidemia; and cancer.
 4. A method for a) increasing energy expenditure of a subject, optionally without impacting the food intake of said subject; or b) increasing satiety in a subject, comprising administering Akkermansia muciniphila or fragments thereof to the subject, wherein the Akkermansia muciniphila is pasteurized.
 5. (canceled)
 6. The method of claim 1, wherein the Akkermansia muciniphila is orally administered.
 7. The method of claim 1, wherein the Akkermansia muciniphila is administered to the subject in an amount from about 1.10⁴ to about 1.10¹² cells, about 1.10⁵ to about 1.10¹¹ cells, or from about 1.10⁶ to about 1.10¹⁰ cells.
 8. The method of claim 1, wherein the Akkermansia muciniphila is administered at least three times a week.
 9. The method of claim 1, wherein the Akkermansia muciniphila is co-administered with another probiotic strain and/or another bacteria and/or microorganisms with beneficial effects and/or with one or more prebiotics.
 10. The method of claim 1, wherein the Akkermansia muciniphila or fragments thereof is administered as a composition according to any one of claims 1 to 9 in association with an excipient.
 11. The method of claim 10, wherein said composition is a nutritional composition.
 12. The method of claim 10, wherein said composition is orally administered.
 13. The method of claim 1, wherein the Akkermansia muciniphila is administered as a pharmaceutical composition in association with a pharmaceutically acceptable vehicle.
 14. (canceled)
 15. A method for weight loss in a subject in need thereof, comprising administering Akkermansia muciniphila or fragments thereof to the subject, wherein the Akkermansia muciniphila is pasteurized.
 16. The method of claim 15, wherein the Akkermansia muciniphila is administered as a cosmetic composition, and wherein the Akkermansia muciniphila is pasteurized. 